Anti-cd33 antibodies and immunoconjugates

ABSTRACT

The invention provides anti-CD33 antibodies and immunoconjugates and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/171,128, filed Jun. 2, 2016, which is a continuation of InternationalApplication No. PCT/US2014/069874, filed Dec. 12, 2014, which claimspriority to U.S. Provisional Application No. 61/916,087, filed Dec. 13,2013; each of which is incorporated by reference herein in its entiretyfor any purpose.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2018-03-09_01146-0032-01US_Sequence_Listing_ST25.txt” created on Mar.9, 2018, which is 81,920 bytes in size. The information in theelectronic format of the sequence listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to anti-CD33 antibodies andimmunoconjugates and methods of using the same.

BACKGROUND

CD33, a member of the sialic acid binding, immunoglobulin-like lectinfamily, is a 67-kDa glycosylated transmembrane protein. It is expressedon most myeloid and monocytic leukemia cells in addition to committedmyelomonocytic and erythroid progenitor cells. It is not seen on theearliest pluripotent stem cells, mature granulocytes, lymphoid cells, ornonhematopoietic cells. See Sabbath et al., J. Clin. Invest. 75:756-56(1985) and Andrews et al., Blood 68:1030-5 (1986). CD33 contains twotyrosine residues on its cytoplasmic tail, each of which is followed byhydrophobic residues similar to the immunoreceptor tyrosine-basedinhibitory motif (ITIM) seen in many inhibitory receptors.

Monoclonal antibody (mAb)-based therapy has become an importanttreatment modality for cancer. Leukemia is well suited to this approachbecause of the accessibility of malignant cells in the blood, bonemarrow, spleen, and lymph nodes and the well-defined immunophenotypes ofthe various lineages and stages of hematopoietic differentiation thatpermit identification of antigenic targets. Most studies for acutemyeloid leukemia (AML) have focused on CD33. Responses with theunconjugated anti-CD33 mAb lintuzumab have had modest single agent andactivity against AML and failed to improve patient outcomes in tworandomized trials when combined with conventional chemotherapy. Theimmunoconjugate gemtuzumab ozogamicin (GO; Mylotarg), an anti-CD33monoclonal antibody conjugated to the antitumor antibioticcalicheamicin, improved survival in a subset of AML patients whencombined with standard chemotherapy, but safety concerns led tomarketing withdrawal in the US. Additionally, three phase I studies ofan anti-CD33-maytansine conjugate (AVE9633; huMy9-6-DM4) in AMLpatients. The maximum tolerated dose (MTD) was determined only in one ofthe phase I studies (administration schedule day 1/8) as the other twostudies were discontinued before reaching the MTD since no signs ofactivity were apparent at doses much higher than the saturating dose.The activity of AVE9633 in the phase I administration schedule day 1/8was modest. Lapusan et al., Invest. New Drugs 30:1121-1131 (2012).

There is a need in the art for safe and effective agents that targetCD33 for the diagnosis and treatment of CD33-associated conditions, suchas cancer. The invention fulfills that need and provides other benefits.

SUMMARY

The invention provides anti-CD33 antibodies and immunoconjugates andmethods of using the same.

In some embodiments, an isolated antibody that binds to CD33 isprovided. In some embodiments, the antibody binds to CD33 and has one ormore of the following characteristics:

-   -   a) binds to recombinant human CD33;    -   b) binds to recombinant cynomolgus monkey CD33;    -   c) binds to endogenous CD33 on the surface of human peripheral        blood mononucleocytes (PBMCs);    -   d) binds to endogenous CD33 on the surface of cynomolgus monkey        PBMCs;    -   e) binds to endogenous CD33 on the surface of a cancer cell;    -   f) binds to endogenous CD33 on the surface of an AML cancer        cell;    -   g) binds to endogenous CD33 on the surface of Molm-13 cells;    -   h) binds to CD33 comprising a R69G mutation;    -   i) binds to CD33 Ig V domain;    -   j) binds to CD33 that is void of N-linked glycosylation at N100;    -   k) binds to CD33 that is void of N-linked glycosylation at N113;    -   1) binds to CD33 comprising an S102A mutation;    -   m) binds to CD33 comprising an S115A mutation;    -   n) does not bind CD33 Ig C2 domain;    -   o) competes for human CD33 binding with My9.6 antibody;    -   p) competes for human CD33 binding with antibody 33H4;    -   q) competes for human CD33 binding with antibody 23E4;    -   r) binds to endogenous human CD33 with a Kd of less than 15 nM,        less than 10 nM, less than 7 nM, less than 5 nM, or less than 3        nM;    -   s) binds to recombinant human CD33 with a Kd of less than 10 nM,        less than 7 nM, less than 5 nM, or less than 3 nM; and/or    -   t) binds to recombinant cynomolgus monkey CD33 with a Kd of less        than 10 nM, less than 7 nM, less than 5 nM, or less than 3 nM,        less than 2 nM, or less than 1 nM.

In some embodiments, an isolated antibody that binds to CD33 isprovided, wherein the antibody comprises (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 112; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 113; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 114; (d) HVR-L1 comprising the amino acidsequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 111; and (f) HVR-L3 comprising the amino acid sequence ofSEQ ID NO:7. In some embodiments, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 115; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 116; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 117; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7. In some embodiments, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:118; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:25; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 111; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.

In some embodiments, an antibody that binds CD33 comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO: 11, SEQ IDNO:20, SEQ ID NO:23, or SEQ ID NO:30; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:21, SEQ ID NO:24,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10, SEQ IDNO:13, SEQ ID NO:22, or SEQ ID NO:25; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, orSEQ ID NO:29; and (f) HVR-L3 comprising the amino acid sequence of SEQID NO:7.

In some embodiments, an antibody that binds CD33 comprises:

(i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:9; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 13; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:21; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:22; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(iv) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(v) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:26; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(vi) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:27; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(vii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:28; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(viii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:29; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(ix) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:30; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(x) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:31; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(xi) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:32; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(xii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:33; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7;

(xiii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:34; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7; or

(xiv) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:35; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In some embodiments, an antibody that binds CD33 comprises:

a) a heavy chain variable region comprising the sequence of SEQ ID NO:66 and a light chain variable region comprising the sequence of SEQ IDNO: 65;

b) a heavy chain variable region comprising the sequence of SEQ ID NO:68 and a light chain variable region comprising the sequence of SEQ IDNO: 67;

c) a heavy chain variable region comprising the sequence of SEQ ID NO:78 and a light chain variable region comprising the sequence of SEQ IDNO: 77;

d) a heavy chain variable region comprising the sequence of SEQ ID NO:80 and a light chain variable region comprising the sequence of SEQ IDNO: 79;

e) a heavy chain variable region comprising the sequence of SEQ ID NO:82 and a light chain variable region comprising the sequence of SEQ IDNO: 81;

f) a heavy chain variable region comprising the sequence of SEQ ID NO:84 and a light chain variable region comprising the sequence of SEQ IDNO: 83;

g) a heavy chain variable region comprising the sequence of SEQ ID NO:86 and a light chain variable region comprising the sequence of SEQ IDNO: 85;

h) a heavy chain variable region comprising the sequence of SEQ ID NO:88 and a light chain variable region comprising the sequence of SEQ IDNO: 87;

i) a heavy chain variable region comprising the sequence of SEQ ID NO:90 and a light chain variable region comprising the sequence of SEQ IDNO: 89;

j) a heavy chain variable region comprising the sequence of SEQ ID NO:92 and a light chain variable region comprising the sequence of SEQ IDNO: 91;

k) a heavy chain variable region comprising the sequence of SEQ ID NO:94 and a light chain variable region comprising the sequence of SEQ IDNO: 93;

l) a heavy chain variable region comprising the sequence of SEQ ID NO:96 and a light chain variable region comprising the sequence of SEQ IDNO: 95;

m) a heavy chain variable region comprising the sequence of SEQ ID NO:98 and a light chain variable region comprising the sequence of SEQ IDNO: 97; or

n) a heavy chain variable region comprising the sequence of SEQ ID NO:100 and a light chain variable region comprising the sequence of SEQ IDNO: 99.

In some embodiments, an isolated antibody that binds to CD33 isprovided. In some embodiments, the antibody binds to CD33 and has one ormore of the following characteristics:

-   -   u) binds to recombinant human CD33;    -   v) binds to recombinant cynomolgus monkey CD33;    -   w) binds to endogenous CD33 on the surface of human peripheral        blood mononucleocytes (PBMCs);    -   x) binds to recombinant human CD33 with a Kd of less than 10 nM,        less than 7 nM, less than 5 nM, less than 3 nM, less than 2 nM,        or less than 1 nM; and/or    -   y) binds to recombinant cynomolgus monkey CD33 with a Kd of less        than 10 nM, less than 7 nM, less than 5 nM, less than 3 nM, less        than 2 nM, or less than 1 nM.

In some embodiments, an isolated antibody that binds to CD33 comprises(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 14; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 15; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16.

In some embodiments, an antibody that binds CD33 comprises:

-   -   z) a heavy chain variable region comprising the sequence of SEQ        ID NO: 70 and a light chain variable region comprising the        sequence of SEQ ID NO: 69;    -   aa) a heavy chain variable region comprising the sequence of SEQ        ID NO: 72 and a light chain variable region comprising the        sequence of SEQ ID NO: 71;    -   bb) a heavy chain variable region comprising the sequence of SEQ        ID NO: 74 and a light chain variable region comprising the        sequence of SEQ ID NO: 73; or    -   cc) a heavy chain variable region comprising the sequence of SEQ        ID NO: 76 and a light chain variable region comprising the        sequence of SEQ ID NO: 75.

In some embodiments, an isolated antibody that binds to CD33 comprises(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In some embodiments, an isolated antibody that binds to CD33 comprises(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:26; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In some embodiments, an isolated antibody that binds to CD33 comprises(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 14; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 15; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a human, humanized, or chimeric antibody.In some embodiments, the antibody is an IgG1, IgG2a or IgG2b antibody.In some embodiments, the antibody is an antibody fragment that bindsCD33. In some embodiments, CD33 is human CD33 has the sequence of SEQ IDNO: 1, with or without a signal sequence (e.g., with or without aminoacids 1-17).

In some embodiments, an isolated nucleic acid encoding an antibodydescribed herein is provided. In some embodiments, a host cellcomprising the nucleic acid is provided. In some embodiments, a methodof producing an antibody comprising culturing the host cell so that theantibody is produced is provided.

In some embodiments, an immunoconjugate comprising an antibody describedherein and a cytotoxic agent is provided. In some embodiments, theimmunoconjugate has the formula Ab-(L-D)p, wherein:

-   -   (a) Ab is the antibody of any one of claim 1 to 15;    -   (b) L is a linker;    -   (c) D is a cytotoxic agent; and    -   (d) p ranges from 1-8.

In some embodiments, the cytotoxic agent is selected from amaytansinoid, a calicheamicin, a pyrrolobenzodiazepine, and anemorubicin derivative. In some embodiments, D is apyrrolobenzodiazepine of Formula A:

-   -   wherein the dotted lines indicate the optional presence of a        double bond between C1 and C2 or C2 and C3;    -   R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR,        ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, and optionally        further selected from halo or dihalo, wherein R^(D) is        independently selected from R, CO₂R, COR, CHO, CO₂H, and halo;    -   R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR,        NH₂, NHR, NRR′, NO₂, Me₃Sn and halo;    -   R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂,        NHR, NRR′, NO₂, Me₃Sn and halo;    -   Q is independently selected from O, S and NH;    -   R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal        cation;    -   R and R′ are each independently selected from optionally        substituted C₁₋₈ alkyl, C₃₋₈ heterocyclyl and C₅₋₂₀ aryl groups,        and optionally in relation to the group NRR′, R and R′ together        with the nitrogen atom to which they are attached form an        optionally substituted 4-, 5-, 6- or 7-membered heterocyclic        ring;    -   R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷        respectively;    -   R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by        one or more heteroatoms and/or aromatic rings that are        optionally substituted; and    -   X and X′ are independently selected from O, S and N(H).

In some embodiments, D has the structure:

-   -   wherein n is 0 or 1.

In some embodiments, D is a nemorubicin derivative. In some embodiments,D has a structure selected from:

In some embodiments, an immunoconjugate comprises a linker that iscleavable by a protease. In some embodiments, an immunoconjugatecomprises a linker that is acid-labile. In some embodiments, the linkercomprises hydrazone.

In some embodiments, an immunoconjugate comprising an antibody describedherein has a formula selected from:

In any of the immunoconjugate embodiments described herein, p rangesfrom 2-5.

In some embodiments, pharmaceutical formulations are provided. In someembodiments, a pharmaceutical formulation comprises an immunoconjugatedescribed herein and a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical formulation comprises an additionaltherapeutic agent.

In some embodiments, methods of treatment are provided. In someembodiments, methods of treating CD33-positive cancers are provided. Insome embodiments, a method of treatment comprises administering to anindividual an effective amount of an immunoconjugate described herein ora pharmaceutical formulation described herein. In some embodiments, theCD33-positive cancer is AML. In some embodiments, the method comprisesadministering an additional therapeutic agent to the individual.

In some embodiments, methods of inhibiting proliferation of aCD33-positive cell are provided. In some embodiments, the methodcomprises exposing the cell to an immunoconjugate described herein underconditions permissive for binding of the immunoconjugate to CD33 on thesurface of the cell, thereby inhibiting proliferation of the cell. Insome embodiments, the cell is an AML cancer cell.

In some embodiments, a method of detecting human CD33 in a biologicalsample is provided. In some embodiments, a method comprises contactingthe biological sample with an anti-CD33 antibody under conditionspermissive for binding of the anti-CD33 antibody to a naturallyoccurring human CD33, and detecting whether a complex is formed betweenthe anti-CD33 antibody and a naturally occurring human CD33 in thebiological sample. In some embodiments, an anti-CD33 antibody is anantibody described herein. In some embodiments, the biological sample isan AML cancer sample.

In some embodiments, a method for detecting a CD33-positive cancer isprovided. In some such embodiments, a method comprises (i) administeringa labeled anti-CD33 antibody to a subject having or suspected of havinga CD33-positive cancer, and (ii) detecting the labeled anti-CD33antibody in the subject, wherein detection of the labeled anti-CD33antibody indicates a CD33-positive cancer in the subject. In someembodiments, an anti-CD33 antibody is an antibody described herein. Insome such embodiments, the labeled anti-CD33 antibody comprises ananti-CD33 antibody conjugated to a positron emitter. In someembodiments, the positron emitter is ⁸⁹Zr.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B shows alignment of the light chain variable region sequences(SEQ ID NOs: 65, 67, 77, and 79, respectively) (A) and heavy chainvariable region sequences (SEQ ID NOs: 66, 68, 78, and 80, respectively)(B) of 7A1, 9C2, 10D3, and 15G15.

FIG. 2A-1-2B-2 shows alignment of the light chain variable regionsequences (SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99,respectively) (A-1 and A-2) and heavy chain variable region sequences(SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, and 100,respectively) (B-1 and B-2) of 15G15, 15G15.33, 15G15.37, 15G15.83,15G15.88, 15G15.7, 15G15.17, 15G15.30, 15G15.31, 15G15.39, and 15G15.84.

FIG. 3A-3B shows alignment of the light chain variable region sequences(SEQ ID NOs: 101, 103, 105, 107, and 109, respectively) (A) and heavychain variable region sequences (SEQ ID NOs: 102, 104, 106, 108, and110, respectively) (B) of 23E4, 27C6, 33F3, 33F9, and 33H4.

FIG. 4A-4B shows alignment of the light chain variable region sequences(SEQ ID NOs: 69, 71, 73, and 75, respectively) (A) and heavy chainvariable region sequences (SEQ ID NOs: 70, 72, 74, and 76, respectively)(B) of 9C3, 9C3.2, 9C3.3, and 9C3.4.

FIG. 5A-5D show species cross-reactivity of anti-CD33 antibodies torecombinant CD33. Anti-CD33 antibody binding to 293 cells expressingrecombinant human (hu) CD33 (A, C) and cynomolgus (cyno) CD33 (B, D) isshown.

FIG. 6A-6D shows anti-CD33 antibody binding to endogenous huCD33expressed in Molm-13 (A, C) and AML (B, D) cells.

FIG. 7A-7D shows species cross-reactivity of anti-CD33 antibodies toendogenous CD33. Anti-CD33 antibody binding to huCD33+(A, C) andcynoCD33+(B, D) myeloid cells.

FIG. 8A-8D shows anti-CD33 antibody epitope binning and comparison toMY9.6 using huCD33 (A-C) and cynoCD33 (D). See e.g., Griffin et al.,LeukRes. 8:521 (1984) regarding MY9.

FIG. 9 shows an exemplary antibody competition experiment betweenanti-CD33 antibody 15G15 and anti-CD33 antibodies 27C6, 9C2, 33F9, 10D3,7A1, 15G15, 23E4, and 33H4 for human CD33 binding.

FIG. 10A-10C shows anti-CD33 antibody 9C3 does not compete withanti-CD33 antibody 15G15.33 and binds to an epitope distinct from15G15.33.

FIG. 11 shows that variants of 9C3 antibody, 9C3.2, 9C3.3, and 9C3.4,have improved binding to huCD33.

FIG. 12A-12D. FIG. 12A shows a schematic of the domain swappedpolypeptide used in the Examples. FIG. 12B-12D shows that anti-CD33antibodies 7A1, 9C2, 10D3 and 15G15 bind to Ig-like V domain of huCD33.

FIG. 13A-13C shows that anti-CD33 antibodies WM53 (A) and 15G15 (B), arecapable of binding to huCD33 Ig-like V domain void of N-linkedglycosylation. In FIG. 13C, the consensus N-glycosylation site sequencesof the Ig-like V domain of huCD33 are boxed, including mutation sitesS102A and S115A. (Because only the Ig-like V domain is shown, thenumbering shown in the figure is different from the numbering of thefull-length CD33.) (huCD33_G69 corresponds to SEQ ID NO: 122, huCD33_R69corresponds to SEQ ID NO: 123).

FIG. 14A-14C shows the wild-type and single nucleotide polymorphism(SNP) sequences of the R69 and G69 Ig-V domain of CD33 (A), and showsthat the binding of the anti-CD33 antibodies, 7A1, 9C2, 10D3, 15G15,23E4, 27C6, 33F9, and 33H4, was not affected by the SNP (r2455069)(e.g., R69G). (huCD33_G69 corresponds to SEQ ID NO: 122, huCD33_R69corresponds to SEQ ID NO: 123).

FIG. 15A-15B shows internalization and in vitro potency of anti-CD33antibodies, 15G15 and 15G15.33 L-D#1.

FIG. 16A-16B shows change in tumor volume (mm³) over time upon treatmentwith 15G15.33 Thio-HC A118C L-D#1 (L-D#1 is made by conjugating thelinker-drug moiety monomethyl-pyridyl disulfide, N10-linkedpyrrolobenzodiazepine

and 15G15.33 Thio-LC V205C L-D#1 at various doses in HL-60 (A) and EOL-1(B) xenografts.

FIG. 17 shows change in tumor volume (mm³) over time upon treatment with15G15 Thio-HC A118C L-D#2 (L-D#2 is made by conjugating the linker-drugmoiety maleimide with acetal linker-PNU

at various concentrations in HL-60 xenografts.

DETAILED DESCRIPTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-CD33 antibody” and “an antibody that binds to CD33”refer to an antibody that is capable of binding CD33 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting CD33. In one embodiment, the extent ofbinding of an anti-CD33 antibody to an unrelated, non-CD33 protein isless than about 10% of the binding of the antibody to CD33 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to CD33 has a dissociation constant (Kd) of ≤1 μM, z≤100 nM,≤10 nM, ≤5 nm, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001nM (e.g., 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ Mto 10⁻¹³ M). In certain embodiments, an anti-CD33 antibody binds to anepitope of CD33 that is conserved among CD33 from different species.

The term “antibody” is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody and that bindsthe antigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia(CML), chronic myelomonocytic leukemia, acute promyelocytic leukemia(APL), chronic myeloproliferative disorder, thrombocytic leukemia,precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), precursorT-cell acute lymphoblastic leukemia (preT-ALL), multiple myeloma (MM),mast cell disease, mast cell leukemia, mast cell sarcoma, myeloidsarcomas, lymphoid leukemia, and undifferentiated leukemia. In someembodiments, the cancer is myeloid leukemia. In some embodiments, thecancer is acute myeloid leukemia (AML).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called β, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Res⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: Clq binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The term “glycosylated forms of CD33” refers to naturally occurringforms of CD33 that are post-translationally modified by the addition ofcarbohydrate residues.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated nucleic acid” refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-CD33 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “CD33,” as used herein, refers to any native, mature CD33 whichresults from processing of a CD33 precursor protein in a cell. The termincludes CD33 from any vertebrate source, including mammals such asprimates (e.g. humans and cynomolgus monkeys) and rodents (e.g., miceand rats), unless otherwise indicated. The term also includes naturallyoccurring variants of CD33, e.g., splice variants or allelic variants.The amino acid sequence of an exemplary human CD33 precursor protein,with signal sequence (with signal sequence, amino acids 1-17) is shownin SEQ ID NO: 1. The amino acid sequence of an exemplary mature humanCD33 is amino acids 18-364 of SEQ ID NO: 1. The amino acid sequence ofan exemplary extracellular domain is amino acids 18-259 of SEQ ID NO: 1.The amino acid sequence of an exemplary Ig-like V-type (Ig V) domain isSEQ ID NO:2. The amino acid sequence of an exemplary Ig-like C2 type (IgC2) domain is SEQ ID NO:3. The amino acid sequence of an exemplarycynomolgus monkey CD33 precursor protein, with signal sequence, is shownin SEQ ID NO:4.

The term “CD33-positive cancer” refers to a cancer comprising cells thatexpress CD33 on their surface. In some embodiments, expression of CD33on the cell surface is determined, for example, using antibodies to CD33in a method such as immunohistochemistry, FACS, etc. Alternatively, CD33mRNA expression is considered to correlate to CD33 expression on thecell surface and can be determined by a method selected from in situhybridization and RT-PCR (including quantitative RT-PCR).

The term “CD33-positive cell” refers to a cell that expresses CD33 onits surface.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₁₂ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 12carbon atoms. A C₁-C₁₂ alkyl group can be unsubstituted or substitutedwith one or more groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₆ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 6 carbonatoms. Representative “C₁-C₆ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, - and n-hexyl;while branched C₁-C₆ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl;unsaturated C₁-C₆ alkyls include, but are not limited to, -vinyl,-allyl, -1-butenyl, -2-butenyl, and -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. A C₁-C₆ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

The term “C₁-C₄ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 4 carbonatoms. Representative “C₁-C₄ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl; while branched C₁-C₄ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl; unsaturated C₁-C₄ alkyls include, but are not limited to,-vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutylenyl. A C₁-C₄ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

“Alkoxy” is an alkyl group singly bonded to an oxygen. Exemplary alkoxygroups include, but are not limited to, methoxy (—OCH₃) and ethoxy(—OCH₂CH₃). A “C₁-C₅ alkoxy” is an alkoxy group with 1 to 5 carbonatoms. Alkoxy groups may can be unsubstituted or substituted with one ormore groups, as described above for alkyl groups.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂). A “C₂-C₈ alkenyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond.

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C≡CH) and propargyl (—CH₂C≡CH). A “C₂-C₈ alkynyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₅-C₂₀ aryl” is an aryl group with 5 to 20 carbon atoms in thecarbocyclic aromatic rings. Examples of C₅-C₂₀ aryl groups include, butare not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₂₀ arylgroup can be substituted or unsubstituted as described above for arylgroups. A “C₅-C₁₄ aryl” is an aryl group with 5 to 14 carbon atoms inthe carbocyclic aromatic rings. Examples of C₅-C₁₄ aryl groups include,but are not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₁₄ arylgroup can be substituted or unsubstituted as described above for arylgroups.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O⁻, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃,—PO₃H₂, —C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —CO₂—, —C(═S)OR, —C(═O)SR,—C(═S)SR, —C(═O)NR₂, —C(═S)NR₂, —C(═NR)NR₂, where each X isindependently a halogen: F, Cl, Br, or I; and each R is independently—H, C₂-C₁₈ alkyl, C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, protecting group orprodrug moiety. Alkylene, alkenylene, and alkynylene groups as describedabove may also be similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 3 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Exemplary heterocycles are described, e.g., in Paquette, Leo A.,“Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York,1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

A “C₃-C₂₀ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. A C₃-C₂₀ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₂₀ heterocyclo” refers to a C₃-C₂₀ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide. In variousembodiments, linkers can comprise one or more amino acid residues, suchas valine, phenylalanine, lysine, and homolysine.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include, but are not limited to,acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ)and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1991, or a lateredition.

II. Compositions and Methods

In one aspect, the invention is based, in part, on antibodies that bindto CD33 and immunoconjugates comprising such antibodies. Antibodies andimmunoconjugates of the invention are useful, e.g., for the diagnosis ortreatment of CD33-positive cancers.

A. Exemplary Anti-CD33 Antibodies

Provided herein are isolated antibodies that bind to CD33. CD33, amember of the sialic acid binding, immunoglobulinlike lectin family, isa 67-kDa glycosylated Type I transmembrane protein, which is expressedon most myeloid and monocytic leukemia cells in addition to committedmyelomonocytic and erythroid progenitor cells

An exemplary naturally occurring human CD33 precursor protein sequence,with signal sequence (amino acids 1-17) is provided in SEQ ID NO: 1, andthe corresponding mature CD33 protein sequence corresponding to aminoacids 18-364 of SEQ ID NO: 1.

In certain embodiments, an anti-CD33 antibody has at least one or moreof the following characteristics, in any combination:

-   -   a) binds to recombinant human CD33;    -   b) binds to recombinant cynomolgus monkey CD33;    -   c) binds to endogenous CD33 on the surface of human peripheral        blood mononucleocytes (PBMCs);    -   d) binds to endogenous CD33 on the surface of cynomolgus monkey        PBMCs;    -   e) binds to endogenous CD33 on the surface of a cancer cell;    -   f) binds to endogenous CD33 on the surface of an AML cancer        cell;    -   g) binds to endogenous CD33 on the surface of Molm-13 cells;    -   h) binds to CD33 comprising a R69G mutation;    -   i) binds to CD33 Ig V domain;    -   j) binds to CD33 that is void of N-linked glycosylation at N100;    -   k) binds to CD33 that is void of N-linked glycosylation at N113;    -   l) binds to CD33 comprising an S102A mutation;    -   m) binds to CD33 comprising an S115A mutation;    -   n) does not bind CD33 Ig C2 domain;    -   o) competes for human CD33 binding with My9.6 antibody;    -   p) competes for human CD33 binding with antibody 33H4;    -   q) competes for human CD33 binding with antibody 23E4;    -   r) binds to endogenous human CD33 with a Kd of less than 15 nM,        less than 10 nM, less than 7 nM, less than 5 nM, or less than 3        nM;    -   s) binds to recombinant human CD33 with a Kd of less than 10 nM,        less than 7 nM, less than 5 nM, or less than 3 nM; and/or    -   t) binds to recombinant cynomolgus monkey CD33 with a Kd of less        than 10 nM, less than 7 nM, less than 5 nM, or less than 3 nM,        less than 2 nM, or less than 1 nM.

In some embodiments, the characteristics of the antibody are determinedas described herein in the Examples below. Nonlimiting exemplary suchantibodies include 7A1, 9C2, 10D3, and 15G15, and variants thereof,described herein. In some embodiments, an antibody that binds CD33 bindsboth recombinant and endogenous human and cynomolgus monkey CD33 andcompetes for human CD33 binding with My9.6, 33H₄, and 23E4. In someembodiments, an antibody that binds CD33 binds both recombinant andendogenous human and cynomolgus monkey CD33 and competes for human CD33binding with My9.6, but has an overlapping but distinct epitope fromMy9.6.

In certain embodiments, an anti-CD33 antibody has at least one or moreof the following characteristics, in any combination:

-   -   a) binds to recombinant human CD33;    -   b) binds to recombinant cynomolgus monkey CD33;    -   c) binds to endogenous CD33 on the surface of human peripheral        blood mononucleocytes (PBMCs);    -   d) binds to recombinant human CD33 with a Kd of less than 10 nM,        less than 7 nM, less than 5 nM, less than 3 nM, less than 2 nM,        or less than 1 nM; and/or    -   e) binds to recombinant cynomolgus monkey CD33 with a Kd of less        than 10 nM, less than 7 nM, less than 5 nM, less than 3 nM, less        than 2 nM, or less than 1 nM.

In some embodiments, the characteristics of the antibody are determinedas described herein in the Examples below. Nonlimiting exemplary suchantibodies include 9C3, and variants thereof, described herein.

Antibody 7A1, 9C2, 10D3, 15G15, 15G15.33, 15G15.37, 15G15.83, 15G15.88,15G15.7, 15G15.17, 15G15.30, 15G15.31, 15G15.39 and Other Embodiments

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, SEQID NO: 11, SEQ ID NO:20, SEQ ID NO:23, and/or SEQ ID NO:30; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:12, SEQ IDNO:21, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, or SEQ ID NO:35; (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6, SEQ ID NO:26, SEQ ID NO:27, SEQID NO:28, or SEQ ID NO:29; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:30; (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:21, SEQ IDNO:24, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:24, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35; and(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:10, SEQ IDNO:13, SEQ ID NO:22, or SEQ ID NO:25. In one embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:10, SEQID NO:13, SEQ ID NO:22, or SEQ ID NO:25. In another embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:25 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:7. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:25, HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:7, and HVR-H2 comprising the aminoacid sequence of SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:21, SEQ ID NO:24,SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:23, orSEQ ID NO:30; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:9, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, or SEQ ID NO:35; and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:22, or SEQID NO:25.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, or SEQ ID NO:29; and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7. In one embodiment, the antibodycomprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5;(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:30,(ii) HVR-H₂ comprising the amino acid sequence of SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:9, SEQ ID NO:12, SEQ IDNO:21, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, or SEQ ID NO:35; and (iii) HVR-H3 comprising an amino acidsequence selected from SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:22, or SEQID NO:25; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, orSEQ ID NO:29, and (c) HVR-L3 comprising the amino acid sequence of SEQID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:20, SEQ ID NO:23, and/or SEQ ID NO:30; (b) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:21,and/or SEQ ID NO:24; (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and/or SEQ ID NO:35; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, and/or SEQ ID NO:29; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:112, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:113, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 114; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 111, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 112; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 113; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 114; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO: 111; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:115, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:116, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 117; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the aminoacid sequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 116; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 117; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:118, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:119, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 111, and (c) HVR-L3 comprising the aminoacid sequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 118; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 119; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 111; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:8, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:9, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 10; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the aminoacid sequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:9; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising theamino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 12, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 13; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the aminoacid sequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 13; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:20, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:21, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:22; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:21; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:22; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:24, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:24, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:26, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:26; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:24, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:27, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:27; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:24, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:28, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:28; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:24, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:29, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:29; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:30, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:24, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:30; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:31, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:31; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:32, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:32; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:33, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:33; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:34, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:34; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising theamino acid sequence of SEQ ID NO:7.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:23, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:35, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:25; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 5, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:7.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:35; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 5; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 6; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:7.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:78, SEQ ID NO:80, SEQID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ IDNO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, or SEQ ID NO:100. Incertain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence ofSEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, and/or SEQ ID NO:100 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-CD33 antibodycomprising that sequence retains the ability to bind to CD33. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:78, SEQID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, and/orSEQ ID NO: 100. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, and/or SEQ ID NO:100. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD33 antibody comprisesthe VH sequence of SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, or SEQ IDNO: 100, including post-translational modifications of that sequence. Ina particular embodiment, the VH comprises one, two or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:8, SEQ ID NO: 11, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:30; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:12,SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, or SEQ ID NO:35; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:25.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:65, SEQ ID NO:67, SEQID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, and/or SEQ ID NO:99. In certain embodiments, a VL sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:77, SEQID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, or SEQ IDNO:99 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:65, SEQ ID NO:67,SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95,SEQ ID NO:97, and/or SEQ ID NO:99. In certain embodiments, a total of 1to 5 amino acids have been substituted, inserted and/or deleted in SEQID NO:65, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ IDNO:93, SEQ ID NO:95, SEQ ID NO:97, and/or SEQ ID NO:99. In certainembodiments, the substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD33antibody comprises the VL sequence of SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, or SEQ ID NO:99, including post-translational modifications ofthat sequence. In a particular embodiment, the VL comprises one, two orthree HVRs selected from (a) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:6, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:66 and SEQ ID NO:65, respectively, including post-translationalmodifications of those sequences. In one embodiment, the antibodycomprises the VH and VL sequences in SEQ ID NO:68 and SEQ ID NO:67,respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:78 and SEQ ID NO:77, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:80 and SEQID NO:79, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:82 and SEQ ID NO:81, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:84 and SEQID NO:83, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:86 and SEQ ID NO:85, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:88 and SEQID NO:87, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:90 and SEQ ID NO:89, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:92 and SEQID NO:91, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:94 and SEQ ID NO:93, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:96 and SEQID NO:95, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:98 and SEQ ID NO:97, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 100 and SEQID NO:99, respectively, including post-translational modifications ofthose sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, or SEQ ID NO: 100 and a VL sequenceof SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81,SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, or SEQ ID NO:99, respectively.

Provided herein are antibodies comprising a light chain variable domaincomprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabatnumbering as depicted in FIGS. 1A and/or 2A and a heavy chain variabledomain comprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according toKabat numbering as depicted in FIGS. 1B and/or 2B. In some embodiments,the antibody comprises a light chain variable domain comprising theHVR1-LC, HVR2-LC and/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LCand/or FR4-LC sequence as depicted in FIGS. 1A and/or 2A. In someembodiments, the antibody comprises a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and/or HVR3-HC sequence, and the FR1-HC,FR2-HC, FR3-HC and/or FR4-HC sequence as depicted in FIGS. 1B and/or 2B.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

Antibody 9C3 and Other Embodiments

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 14; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 15; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:16.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 18; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO: 19. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO: 19 and HVR-L3comprising the amino acid sequence of SEQ ID NO: 16. In a furtherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO: 19, HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 16, and HVR-H2 comprising the amino acid sequence of SEQ IDNO: 18. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 18; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:19.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 15; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 14; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:15; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:16.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 17, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 18, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 19; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 14, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 15, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 16.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 14; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 15; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, and/or SEQ IDNO:76. In certain embodiments, a VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acidsequence of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, and/or SEQ IDNO:76 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, and/or SEQ ID NO:76. In certain embodiments, a total of 1to 5 amino acids have been substituted, inserted and/or deleted in SEQID NO:70, SEQ ID NO:72, SEQ ID NO:74, and/or SEQ ID NO:76. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-CD33 antibodycomprises the VH sequence of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74,or SEQ ID NO:76, including post-translational modifications of thatsequence. In a particular embodiment, the VH comprises one, two or threeHVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQID NO: 17, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:18, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:19.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:69, SEQ ID NO:71, SEQID NO:73, and/or SEQ ID NO:75. In certain embodiments, a VL sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO:69, SEQ ID NO:71, SEQID NO:73, and/or SEQ ID NO:75 contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-CD33 antibody comprising that sequence retains theability to bind to CD33. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, and/or SEQ ID NO:75. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73,and/or SEQ ID NO:75. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-CD33 antibody comprises the VL sequence ofSEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, or SEQ ID NO:75, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 15; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:70 and SEQ ID NO:69, respectively, including post-translationalmodifications of those sequences. In one embodiment, the antibodycomprises the VH and VL sequences in SEQ ID NO:72 and SEQ ID NO:71,respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:74 and SEQ ID NO:73, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:76 and SEQID NO:75, respectively, including post-translational modifications ofthose sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, and SEQ ID NO:76 and a VL sequence ofSEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, and SEQ ID NO:75,respectively.

Provided herein are antibodies comprising a light chain variable domaincomprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabatnumbering as depicted in FIG. 4A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 4B. In some embodiments, the antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 4A. In some embodiments, the antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 4B.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

Antibody 23E4 and Other Embodiments

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:40; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:41; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:36; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:37; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:38.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:40; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:41. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:41. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:41 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:38. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:41, HVR-L3 comprising the amino acid sequence of SEQ ID NO:38, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:40. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:39; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:40; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:41.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:36; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:37; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:38. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:36; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:37; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:38.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:39, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:40, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:41; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:36, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:37, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:38.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:40; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:41; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:36; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:37; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:38.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 102. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 102 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD33 antibody comprising that sequenceretains the ability to bind to CD33. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 102. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 102. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD33antibody comprises the VH sequence of SEQ ID NO: 102, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:39, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:40, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:41.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:101. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ IDNO:101 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:101. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:101. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD33 antibody comprisesthe VL sequence of SEQ ID NO: 101, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:36; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:37; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:38.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO: 102 and SEQ ID NO: 101, respectively, includingpost-translational modifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ ID NO:102 and a VL sequence of SEQ ID NO:101.

Provided herein are 23E4 antibodies comprising a light chain variabledomain comprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according toKabat numbering as depicted in FIG. 3A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 3B. In some embodiments, the 23E4 antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 3A. In some embodiments, the 23E4 antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 3B.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

Antibody 27C6 and Other Embodiments

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:45; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:46; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:47; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:42; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:43; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:44.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:45; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:46; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:47. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:47. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:47 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:44. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:47, HVR-L3 comprising the amino acid sequence of SEQ ID NO:44, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:46. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:45; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:46; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:47.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:42; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:43; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:44. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:42; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:43; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:44.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:45, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:46, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:47; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:42, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:43, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:44.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:45; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:46; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:47; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:42; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:43; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:44.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 104. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 104 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD33 antibody comprising that sequenceretains the ability to bind to CD33. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 104. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 104. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD33antibody comprises the VH sequence of SEQ ID NO: 104, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:45, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:46, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:47.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 103. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:103 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 103. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 103. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD33 antibody comprisesthe VL sequence of SEQ ID NO: 103, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:42; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:43; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:44.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO: 104 and SEQ ID NO: 103, respectively, includingpost-translational modifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ ID NO:104 and a VL sequence of SEQ ID NO:103.

Provided herein are 27C6 antibodies comprising a light chain variabledomain comprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according toKabat numbering as depicted in FIG. 3A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 3B. In some embodiments, the 27C6 antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 3A. In some embodiments, the antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 3B.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

Antibody 33F3 and Other Embodiments

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:45; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:51; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:52; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:48; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:49; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:50.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:45; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:51; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:52. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:52. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:52 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:50. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:52, HVR-L3 comprising the amino acid sequence of SEQ ID NO:50, andHVR-H2 comprising the amino acid sequence of SEQ ID NO: 51. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:45; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:51; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:52.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:48; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:49; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:50. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:48; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:49; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:50.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:45, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:51, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 52; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:48, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:49, and (c) HVR-L3 comprising the aminoacid sequence of SEQ ID NO:50.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:45; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:51; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:52; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:48; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:49; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:50.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 106. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 106 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD33 antibody comprising that sequenceretains the ability to bind to CD33. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 106. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 106. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD33antibody comprises the VH sequence of SEQ ID NO: 106, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:45, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:51, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 52.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 105. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:105 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 105. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 105. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD33 antibody comprisesthe VL sequence of SEQ ID NO: 105, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:48; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:49; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:50.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO: 106 and SEQ ID NO: 105, respectively, includingpost-translational modifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ ID NO:106 and a VL sequence of SEQ ID NO:105.

Provided herein are 33F3 antibodies comprising a light chain variabledomain comprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according toKabat numbering as depicted in FIG. 3A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 3B. In some embodiments, the 33F3 antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 3A. In some embodiments, the 33F3 antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 3B.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

Antibody 33F9 and Other Embodiments

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:56; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:57; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:58; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:53; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:54; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:55.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:56; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:57; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:58. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:58. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:58 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:55. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:58, HVR-L3 comprising the amino acid sequence of SEQ ID NO:55, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:57. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:56; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:57; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:58.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:53; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:54; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:55. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:53; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:54; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:55.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 56, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 57, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:58; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:53, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:54, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:55.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:56; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:57; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:58; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:53; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:54; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:55.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 108. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 108 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD33 antibody comprising that sequenceretains the ability to bind to CD33. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 108. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 108. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD33antibody comprises the VH sequence of SEQ ID NO: 108, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:56, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:57, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:58.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 107. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:107 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 107. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 107. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD33 antibody comprisesthe VL sequence of SEQ ID NO: 107, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:53; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:54; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:55.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO: 108 and SEQ ID NO: 107, respectively, includingpost-translational modifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ ID NO:108 and a VL sequence of SEQ ID NO:107.

Provided herein are 33F9 antibodies comprising a light chain variabledomain comprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according toKabat numbering as depicted in FIG. 3A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 3B. In some embodiments, the 33F9 antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 3A. In some embodiments, the 33F9 antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 3B.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

Antibody 33H4 and Other Embodiments

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:62; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:64; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:59; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:60; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:61.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:62; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:63; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:64. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:64. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:64 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:61. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:64, HVR-L3 comprising the amino acid sequence of SEQ ID NO:61, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:63. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:62; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:63; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:64.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:59; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:60; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:61. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:59; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:60; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:61.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:62, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:63, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:64; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:59, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:60, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:61.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:62; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:63; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:64; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:59; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:60; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:61.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 110. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 110 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD33 antibody comprising that sequenceretains the ability to bind to CD33. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 110. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 110. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD33antibody comprises the VH sequence of SEQ ID NO: 110, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:62, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:63, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:64.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 109. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:109 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 109. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 109. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD33 antibody comprisesthe VL sequence of SEQ ID NO: 109, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:59; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:60; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:61.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO: 110 and SEQ ID NO: 109, respectively, includingpost-translational modifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ ID NO:110 and a VL sequence of SEQ ID NO:109.

Provided herein are 33H4 antibodies comprising a light chain variabledomain comprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according toKabat numbering as depicted in FIG. 3A and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 3B. In some embodiments, the 33H4 antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 3A. In some embodiments, the 33H4 antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 3B.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 NM, ≤100 nM, ≤50 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM, and optionally is ≥10⁻¹³ M. (e.g. 10⁻⁸ M orless, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵J]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 pd/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO M spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthin, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for CD33 and the other is for any other antigen. Incertain embodiments, one of the binding specificities is for CD33 andthe other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certainembodiments, bispecific antibodies may bind to two different epitopes ofCD33. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express CD33. Bispecific antibodies can beprepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to CD33 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout +3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq bindingassays may also be carried out to confirm that the antibody is unable tobind Clq and hence lacks CDC activity. See, e.g., Clq and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) Clq binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-CD33 antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-CD33 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-CD33 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-CD33 antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, BIACore®, FACS,or Western blot.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies described herein forbinding to CD33. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by an antibody described herein. Detailed exemplary methods formapping an epitope to which an antibody binds are provided in Morris(1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol.66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized CD33 is incubated in asolution comprising a first labeled antibody that binds to CD33 (e.g.,any of the antibodies described herein) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to CD33. The second antibody may be present in a hybridomasupernatant. As a control, immobilized CD33 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to CD33, excess unbound antibody is removed, and theamount of label associated with immobilized CD33 is measured. If theamount of label associated with immobilized CD33 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to CD33. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD33antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes(i.e., a radioconjugate).

Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and, in some embodiments intracellular accumulation therein,where systemic administration of unconjugated drugs may result inunacceptable levels of toxicity to normal cells (Polakis P. (2005)Current Opinion in Pharmacology 5:382-387).

Antibody-drug conjugates (ADC) are targeted chemotherapeutic moleculeswhich combine properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells(Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), therebyenhancing the therapeutic index by maximizing efficacy and minimizingoff-target toxicity (Carter, P. J. and Senter P. D. (2008) The CancerJour. 14(3):154-169; Chari, R. V. (2008) Acc. Chem. Res. 41:98-107.

The ADC compounds of the invention include those with anticanceractivity. In some embodiments, the ADC compounds include an antibodyconjugated, i.e. covalently attached, to the drug moiety. In someembodiments, the antibody is covalently attached to the drug moietythrough a linker. The antibody-drug conjugates (ADC) of the inventionselectively deliver an effective dose of a drug to tumor tissue wherebygreater selectivity, i.e. a lower efficacious dose, may be achievedwhile increasing the therapeutic index (“therapeutic window”).

The drug moiety (D) of the antibody-drug conjugates (ADC) may includeany compound, moiety or group that has a cytotoxic or cytostatic effect.Drug moieties may impart their cytotoxic and cytostatic effects bymechanisms including but not limited to tubulin binding, DNA binding orintercalation, and inhibition of RNA polymerase, protein synthesis,and/or topoisomerase. Exemplary drug moieties include, but are notlimited to, a maytansinoid, dolastatin, auristatin, calicheamicin,pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives,PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane,trichothecene, CC1065, camptothecin, elinafide, and stereoisomers,isosteres, analogs, and derivatives thereof that have cytotoxicactivity. Nonlimiting examples of such immunoconjugates are discussed infurther detail below.

1. Exemplary Antibody-Drug Conjugates

An exemplary embodiment of an antibody-drug conjugate (ADC) compoundcomprises an antibody (Ab) which targets a tumor cell, a drug moiety(D), and a linker moiety (L) that attaches Ab to D. In some embodiments,the antibody is attached to the linker moiety (L) through one or moreamino acid residues, such as lysine and/or cysteine.

An exemplary ADC has Formula I:

Ab-(L-D)_(p)  I

where p is 1 to about 20. In some embodiments, the number of drugmoieties that can be conjugated to an antibody is limited by the numberof free cysteine residues. In some embodiments, free cysteine residuesare introduced into the antibody amino acid sequence by the methodsdescribed herein. Exemplary ADC of Formula I include, but are notlimited to, antibodies that have 1, 2, 3, or 4 engineered cysteine aminoacids (Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In someembodiments, one or more free cysteine residues are already present inan antibody, without the use of engineering, in which case the existingfree cysteine residues may be used to conjugate the antibody to a drug.In some embodiments, an antibody is exposed to reducing conditions priorto conjugation of the antibody in order to generate one or more freecysteine residues.

a) Exemplary Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more drug moieties (D) to an antibody (Ab) to forman antibody-drug conjugate (ADC) of Formula I. In some embodiments,antibody-drug conjugates (ADC) can be prepared using a Linker havingreactive functionalities for covalently attaching to the drug and to theantibody. For example, in some embodiments, a cysteine thiol of anantibody (Ab) can form a bond with a reactive functional group of alinker or a drug-linker intermediate to make an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linker components are knownin the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula II:

-A_(a)-W_(w)-Y_(y)  II

wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W isan “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacerunit”, and y is 0, 1, or 2; and Ab, D, and p are defined as above forFormula I. Exemplary embodiments of such linkers are described in U.S.Pat. No. 7,498,298, which is expressly incorporated herein by reference.

In some embodiments, a linker component comprises a “stretcher unit”that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, a linker component comprises an “amino acid unit”.In some such embodiments, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784).Exemplary amino acid units include, but are not limited to, dipeptides,tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptidesinclude, but are not limited to, valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline. Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

In some embodiments, a linker component comprises a “spacer” unit thatlinks the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005)15:1087-1103). In some embodiments, the spacer unit isp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro, or -cyno;m is an integer ranging from 0 to 4; and p ranges from 1 to about 20. Insome embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. In some embodiments, spacers can be used thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Linkage of a drug to the α-carbon of a glycineresidue is another example of a self-immolative spacer that may beuseful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al (2002) Bioorganic& Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic& Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase themolar ratio of drug to antibody, i.e. loading, which is related to thepotency of the ADC. Thus, where an antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Nonlimiting exemplary linkers are shown below in the context of an ADCof Formula I:

Further nonlimiting exemplary ADCs include the structures:

where X is:

Y is:

each R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schroder and K. Liibke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃ ⁻) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula I. Insome such embodiments, the antibody comprises more than one (linkerportion) a substituents, such that more than one drug is coupled to theantibody in the ADC of Formula I.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following linker reagents: bis-maleimido-trioxyethy lene glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(ε-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SIAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al (2002) J.Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch etal (1996) Bioconjugate Chem. 7:180-186; U.S. Pat. No. 6,214,345; WO02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; andWO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.

b) Exemplary Drug Moieties

(1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are derivatives ofmaytansine, and are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos.4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Certain maytansinoids suitable for use as maytansinoid drug moieties areknown in the art and can be isolated from natural sources according toknown methods or produced using genetic engineering techniques (see,e.g., Yu et al (2002) PNAS 99:7968-7973). Maytansinoids may also beprepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include, but are not limited to,those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat.No. 4,256,746) (prepared, for example, by lithium aluminum hydridereduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared, forexample, by demethylation using Streptomyces or Actinomyces ordechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (—OCOR),+/−dechloro (U.S. Pat. No. 4,294,757) (prepared, for example, byacylation using acyl chlorides), and those having modifications at otherpositions of the aromatic ring.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, forexample, by the reaction of maytansinol with H₂S or P2S5);C-14-alkoxymethyl(demethoxy/CH₂OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy/acyloxy(U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion ofmaytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and4,315,929) (for example, isolated from Trewia nudlflora);C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, forexample, by the demethylation of maytansinol by Streptomyces); and4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by thetitanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinoid compounds are useful as the linkageposition. For example, an ester linkage may be formed by reaction with ahydroxyl group using conventional coupling techniques. In someembodiments, the reaction may occur at the C-3 position having ahydroxyl group, the C-14 position modified with hydroxymethyl, the C-15position modified with a hydroxyl group, and the C-20 position having ahydroxyl group. In some embodiments, the linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Maytansinoid drug moieties include those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid drug moiety to a linker of an ADC. Each R mayindependently be H or a C₁-C₆ alkyl. The alkylene chain attaching theamide group to the sulfur atom may be methanyl, ethanyl, or propyl,i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020;Chari et al (1992) Cancer Res. 52:127-131; Liu et al (1996) Proc. Natl.Acad. Sci USA 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe ADC of the invention, i.e. any combination of R and S configurationsat the chiral carbons (U.S. Pat. No. 7,276,497; U.S. Pat. No. 6,913,748;U.S. Pat. No. 6,441,163; U.S. Pat. No. 633,410 (RE39151); U.S. Pat. No.5,208,020; Widdison et al (2006) J. Med. Chem. 49:4392-4408, which areincorporated by reference in their entirety). In some embodiments, themaytansinoid drug moiety has the following stereochemistry:

Exemplary embodiments of maytansinoid drug moieties include, but are notlimited to, DM1; DM3; and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody-drug conjugate.

Other exemplary maytansinoid antibody-drug conjugates have the followingstructures and abbreviations (wherein Ab is antibody and p is 1 to about20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is1 to 4):

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20. In someembodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent EP 0425 235 B1, the disclosures of which are hereby expressly incorporatedby reference. See also Liu et al. Proc. Natl. Acad. Sci. USA93:8618-8623 (1996); and Chari et al. Cancer Research 52:127-131 (1992).

In some embodiments, antibody-maytansinoid conjugates may be prepared bychemically linking an antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (thedisclosure of which is hereby expressly incorporated by reference). Insome embodiments, ADC with an average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody. In some instances, even one molecule oftoxin/antibody is expected to enhance cytotoxicity over the use of nakedantibody.

Exemplary linking groups for making antibody-maytansinoid conjugatesinclude, for example, those described herein and those disclosed in U.S.Pat. No. 5,208,020; EP Patent 0 425 235 B1; Chari et al. Cancer Research52:127-131 (1992); US 2005/0276812 A1; and US 2005/016993 A1, thedisclosures of which are hereby expressly incorporated by reference.

(2) Auristatins and Dolastatins

Drug moieties include dolastatins, auristatins, and analogs andderivatives thereof (U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;U.S. Pat. No. 5,767,237; U.S. Pat. No. 6,124,431). Auristatins arederivatives of the marine mollusk compound dolastatin-10. While notintending to be bound by any particular theory, dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin/auristatindrug moiety may be attached to the antibody through the N (amino)terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO02/088172; Doronina et al (2003) Nature Biotechnology 21(7):778-784;Francisco et al (2003) Blood 102(4): 1458-1465).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties D_(E) and D_(F), disclosed in U.S.Pat. No. 7,498,298 and U.S. Pat. No. 7,659,241, the disclosures of whichare expressly incorporated by reference in their entirety:

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:

R² is selected from H and C₁-C₈ alkyl;

R³ is selected from H, C₁-C₈ alkyl, C3-C carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6;

R⁶ is selected from H and C₁-C₈ alkyl;

R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);

R⁹ is selected from H and C₁-C₈ alkyl;

R¹⁰ is selected from aryl or C₃-C₈ heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;

R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂; m is an integer ranging from1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or C₁-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;

each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;

R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and

n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is —H, andR⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(F) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

Other exemplary embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Nonlimiting exemplary embodiments of ADC of Formula I comprising MMAE orMMAF and various linker components have the following structures andabbreviations (wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit”is a valine-citrulline dipeptide; and “S” is a sulfur atom:

Nonlimiting exemplary embodiments of ADCs of Formula I comprising MMAFand various linker components further include Ab-MC-PAB-MMAF andAb-PAB-MMAF. Immunoconjugates comprising MMAF attached to an antibody bya linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker (Doronina et al.(2006) Bioconjugate Chem. 17:114-124). In some such embodiments, drugrelease is believed to be effected by antibody degradation in the cell.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to a liquidphase synthesis method (see, e.g., E. Schroder and K. Liibke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press).Auristatin/dolastatin drug moieties may, in some embodiments, beprepared according to the methods of: U.S. Pat. No. 7,498,298; U.S. Pat.No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al(1996) J. Chem. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In some embodiments, auristatin/dolastatin drug moieties of formulasD_(E) such as MMAE, and D_(F), such as MMAF, and drug-linkerintermediates and derivatives thereof, such as MC-MMAF, MC-MMAE,MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methodsdescribed in U.S. Pat. No. 7,498,298; Doronina et al. (2006)Bioconjugate Chem. 17:114-124; and Doronina et al. (2003) Nat. Biotech.21:778-784 and then conjugated to an antibody of interest.

(3) Calicheamicin

In some embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics, and analogues thereof, are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations (Hinman etal., (1993) Cancer Research 53:3336-3342; Lode et al., (1998) CancerResearch 58:2925-2928). Calicheamicin has intracellular sites of actionbut, in certain instances, does not readily cross the plasma membrane.Therefore, cellular uptake of these agents through antibody-mediatedinternalization may, in some embodiments, greatly enhances theircytotoxic effects. Nonlimiting exemplary methods of preparingantibody-drug conjugates with a calicheamicin drug moiety are described,for example, in U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,714,586; U.S.Pat. No. 5,739,116; and U.S. Pat. No. 5,767,285.

(4) Pyrrolobenzodiazepines

In some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). Insome embodiments, PDB dimers recognize and bind to specific DNAsequences. The natural product anthramycin, a PBD, was first reported in1965 (Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5793-5795;Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5791-5793). Since then,a number of PBDs, both naturally-occurring and analogues, have beenreported (Thurston, et al., (1994) Chem. Rev. 1994, 433-465 includingdimers of the tricyclic PBD scaffold (U.S. Pat. No. 6,884,799; U.S. Pat.No. 7,049,311; U.S. Pat. No. 7,067,511; U.S. Pat. No. 7,265,105; U.S.Pat. No. 7,511,032; U.S. Pat. No. 7,528,126; U.S. Pat. No. 7,557,099).Without intending to be bound by any particular theory, it is believedthat the dimer structure imparts the appropriate three-dimensional shapefor isohelicity with the minor groove of B-form DNA, leading to a snugfit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986) Acc. Chem.Res., 19:230-237). Dimeric PBD compounds bearing C2 aryl substituentshave been shown to be useful as cytotoxic agents (Hartley et al (2010)Cancer Res. 70(17):6849-6858; Antonow (2010) J. Med. Chem.53(7):2927-2941; Howard et al (2009) Bioorganic and Med. Chem. Letters19(22):6463-6466).

In some embodiments, PBD compounds can be employed as prodrugs byprotecting them at the N10 position with a nitrogen protecting groupwhich is removable in vivo (WO 00/12507; WO 2005/023814).

PBD dimers have been conjugated to antibodies and the resulting ADCshown to have anti-cancer properties (US 2010/0203007). Nonlimitingexemplary linkage sites on the PBD dimer include the five-memberedpyrrolo ring, the tether between the PBD units, and the N10-C11 iminegroup (WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431;US 2011/0256157; WO 2011/130598).

Nonlimiting exemplary PBD dimer components of ADCs are of Formula A:

and salts and solvates thereof, wherein:

the wavy line indicates the covalent attachment site to the linker;

the dotted lines indicate the optional presence of a double bond betweenC1 and C2 or C2 and C3;

R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR, ═CH—R^(D),═C(R^(D))₂, O—SO₂—R, CO₂R and COR, and optionally further selected fromhalo or dihalo, wherein R^(D) is independently selected from R, CO₂R,COR, CHO, CO₂H, and halo;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, NO₂, Me₃Sn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo;

Q is independently selected from O, S and NH;

R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal cation;

R and R′ are each independently selected from optionally substitutedC₁₋₈ alkyl, C₁₋₁₂ alkyl, C₃₋₈ heterocyclyl, C₃₋₂₀ heterocycle, and C₅₋₂₀aryl groups, and optionally in relation to the group NRR′, R and R′together with the nitrogen atom to which they are attached form anoptionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively;

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g.benzene or pyridine, which rings are optionally substituted; and

X and X′ are independently selected from O, S and N(H).

In some embodiments, R and R′ are each independently selected fromoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocycle, and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring.

In some embodiments, R⁹ and R¹⁹ are H.

In some embodiments, R⁶ and R¹⁶ are H.

In some embodiments, R⁷ are R¹⁷ are both OR^(7A), where R^(7A) isoptionally substituted C₁₋₄ alkyl. In some embodiments, R^(7A) is Me. Insome embodiments, R^(7A) is is Ch₂Ph, where Ph is a phenyl group.

In some embodiments, X is O.

In some embodiments, R¹¹ is H.

In some embodiments, there is a double bond between C2 and C3 in eachmonomer unit.

In some embodiments, R² and R¹² are independently selected from H and R.In some embodiments, R² and R¹² are independently R. In someembodiments, R² and R¹² are independently optionally substituted C₅₋₂₀aryl or C₅₋₇ aryl or C₈₋₁₀ aryl. In some embodiments, R² and R¹² areindependently optionally substituted phenyl, thienyl, napthyl, pyridyl,quinolinyl, or isoquinolinyl. In some embodiments, R² and R¹² areindependently selected from ═O, ═CH₂, ═CH—R^(D), and ═C(R^(D))₂. In someembodiments, R² and R¹² are each ═CH₂. In some embodiments, R² and R¹²are each H. In some embodiments, R² and R¹² are each ═O.

In some embodiments, R² and R¹² are each ═CF₂. In some embodiments, R²and/or R¹² are independently ═C(R^(D))₂. In some embodiments, R² and/orR¹² are independently ═CH—R^(D).

In some embodiments, when R² and/or R¹² is ═CH—R^(D), each group mayindependently have either configuration shown below:

In some embodiments, a ═CH—R^(D) is in configuration (I).

In some embodiments, R¹¹ is a C3 alkylene group or a C5 alkylene group.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(I):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(II):

wherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(III):

wherein R^(E) and R^(E″) are each independently selected from H orR^(D), wherein R^(D) is defined as above; and wherein n is 0 or 1.

In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, R^(E) and/or R^(E″) is H. In some embodiments, R^(E) andR^(E″) are H. In some embodiments, R^(E) and/or R^(E″) is R^(D), whereinR^(D) is optionally substituted C₁₋₁₂ alkyl. In some embodiments, R^(E)and/or R^(E″) is R^(D), wherein R^(D) is methyl.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(IV):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may bethe same or different; andwherein n is 0 or 1.

In some embodiments, an exemplary PBD dimer component of an ADC has thestructure of Formula A(V):

wherein Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl; wherein Ar¹ and Ar² may be the same or different; and

wherein n is 0 or 1.

In some embodiments, Ar¹ and Ar² are each independently selected fromoptionally substituted phenyl, furanyl, thiophenyl and pyridyl. In someembodiments, Ar¹ and Ar² are each independently optionally substitutedphenyl. In some embodiments, Ar¹ and Ar² are each independentlyoptionally substituted thien-2-yl or thien-3-yl. In some embodiments,Ar¹ and Ar² are each independently optionally substituted quinolinyl orisoquinolinyl. The quinolinyl or isoquinolinyl group may be bound to thePBD core through any available ring position. For example, thequinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl,quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. In someembodiments, the quinolinyl is selected from quinolin-3-yl andquinolin-6-yl. The isoquinolinyl may be isoquinolin-1-yl,isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl,isoquinolin-7-yl and isoquinolin-8-yl. In some embodiments, theisoquinolinyl is selected from isoquinolin-3-yl and isoquinolin-6-yl.

Further nonlimiting exemplary PBD dimer components of ADCs are ofFormula B:

and salts and solvates thereof, wherein:

the wavy line indicates the covalent attachment site to the linker;

the wavy line connected to the OH indicates the S or R configuration;

R^(V1) and R^(V2) are independently selected from H, methyl, ethyl andphenyl (which phenyl may be optionally substituted with fluoro,particularly in the 4 position) and C₅₋₆ heterocyclyl; wherein R^(V1)and R^(V2) may be the same or different; and

n is 0 or 1.

In some embodiments, R^(V1) and R^(V2) are independently selected fromH, phenyl, and 4-fluorophenyl.

In some embodiments, a linker may be attached at one of various sites ofthe PBD dimer drug moiety, including the N10 imine of the B ring, theC-2 endo/exo position of the C ring, or the tether unit linking the Arings (see structures C(I) and C(II) below).

Nonlimiting exemplary PBD dimer components of ADCs include Formulas C(I)and C(II):

Formulas C(I) and C(II) are shown in their N10-C11 imine form. ExemplaryPBD drug moieties also include the carbinolamine and protectedcarbinolamine forms as well, as shown in the table below:

Imine Carbinolamine Protected Carbinolaminewherein:

X is CH₂ (n=1 to 5), N, or O;

Z and Z′ are independently selected from OR and NR₂, where R is aprimary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;

R₁, R′₁, R₂ and R′₂ are each independently selected from H, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀ aryl (including substituted aryls),C₅₋₂₀ heteroaryl groups, —NH₂, —NHMe, —OH, and —SH, where, in someembodiments, alkyl, alkenyl and alkynyl chains comprise up to 5 carbonatoms;

R₃ and R′₃ are independently selected from H, OR, NHR, and NR₂, where Ris a primary, secondary or tertiary alkyl chain containing 1 to 5 carbonatoms;

R₄ and R′₄ are independently selected from H, Me, and OMe;

R₅ is selected from C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₅₋₂₀aryl (including aryls substituted by halo, nitro, cyano, alkoxy, alkyl,heterocyclyl) and C₅₋₂₀ heteroaryl groups, where, in some embodiments,alkyl, alkenyl and alkynyl chains comprise up to 5 carbon atoms;

R₁₁ is H, C₁-C₈ alkyl, or a protecting group (such as acetyl,trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ),9-fluorenylmethylenoxycarbonyl (Fmoc), or a moiety comprising aself-immolating unit such as valine-citrulline-PAB);

R₁₂ is is H, C₁-C₈ alkyl, or a protecting group;

wherein a hydrogen of one of R₁, R′₁, R₂, R′₂, R₅, or R₁₂ or a hydrogenof the —OCH₂CH₂(X)_(n)CH₂CH₂O— spacer between the A rings is replacedwith a bond connected to the linker of the ADC.

Exemplary PDB dimer portions of ADC include, but are not limited to (thewavy line indicates the site of covalent attachment to the linker):

Nonlimiting exemplary embodiments of ADCs comprising PBD dimers have thefollowing structures:

wherein:

n is 0 to 12. In some embodiments, n is 2 to 10. In some embodiments, nis 4 to 8. In some embodiments, n is selected from 4, 5, 6, 7, and 8.

A further non-limiting exemplary ADC comprising a PBD dimer may be madeby conjugating a monomethyl pyridyl disulfide, N10-linked PBD (shownbelow) to an antibody:

The linkers of PBD dimer-val-cit-PAB-Ab and the PBD dimer-Phe-Lys-PAB-Abare protease cleavable, while the linker of PBD dimer-maleimide-acetalis acid-labile.

PBD dimers and ADC comprising PBD dimers may be prepared according tomethods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598.

(5) Anthracyclines

In some embodiments, an ADC comprising anthracycline. Anthracyclines areantibiotic compounds that exhibit cytotoxic activity. While notintending to be bound by any particular theory, studies have indicatedthat anthracyclines may operate to kill cells by a number of differentmechanisms, including: 1) intercalation of the drug molecules into theDNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis;2) production by the drug of free radicals which then react withcellular macromolecules to cause damage to the cells, and/or 3)interactions of the drug molecules with the cell membrane (see, e.g., C.Peterson et al., “Transport And Storage Of Anthracycline In ExperimentalSystems And Human Leukemia” in Anthracycline Antibiotics In CancerTherapy; N. R. Bachur, “Free Radical Damage” id. at pp. 97-102). Becauseof their cytotoxic potential anthracyclines have been used in thetreatment of numerous cancers such as leukemia, breast carcinoma, lungcarcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P. H-Wiernik,in Anthracycline: Current Status And New Developments p 11).

Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin,idarubicin, daunomycin, nemorubicin, and derivatives thereof.Immunoconjugates and prodrugs of daunorubicin and doxorubicin have beenprepared and studied (Kratz et al (2006) Current Med. Chem. 13:477-523;Jeffrey et al (2006) Bioorganic & Med. Chem. Letters 16:358-362; Torgovet al (2005) Bioconj. Chem. 16:717-721; Nagy et al (2000) Proc. Natl.Acad. Sci. USA 97:829-834; Dubowchik et al (2002) Bioorg. & Med. Chem.Letters 12:1529-1532; King et al (2002) J. Med. Chem. 45:4336-4343; EP0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugateBR96-doxorubicin reacts specifically with the tumor-associated antigenLewis-Y and has been evaluated in phase I and II studies (Saleh et al(2000) J. Clin. Oncology 18:2282-2292; Ajani et al (2000) Cancer Jour.6:78-81; Tolcher et al (1999) J. Clin. Oncology 17:478-484).

PNU-159682 is a potent metabolite (or derivative) of nemorubicin(Quintieri, et al. (2005) Clinical Cancer Research 11(4): 1608-1617).Nemorubicin is a semisynthetic analog of doxorubicin with a2-methoxymorpholino group on the glycoside amino of doxorubicin and hasbeen under clinical evaluation (Grandi et al (1990) Cancer Treat. Rev.17:133; Ripamonti et al (1992) Brit. J. Cancer 65:703;), including phaseII/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedingsof the American Society for Clinical Oncology 22, Abs1448; Quintieri(2003) Proceedings of the American Association of Cancer Research,44:1st Ed, Abs 4649; Pacciarini et al (2006) Jour. Clin. Oncology24:14116).

A nonlimiting exemplary ADC comprising nemorubicin or nemorubicinderivatives is shown in Formula Ia:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R2 is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;

L₁ and Z together are a linker (L) as described herein;

T is an antibody (Ab) as described herein; and

m is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R₂ are both methoxy (—OMe).

A further nonlimiting exemplary ADC comprising nemorubicin ornemorubicin derivatives is shown in Formula Ib:

wherein R₁ is hydrogen atom, hydroxy or methoxy group and R₂ is a C₁-C₅alkoxy group, or a pharmaceutically acceptable salt thereof;

L2 and Z together are a linker (L) as described herein;

T is an antibody (Ab) as described herein; and

m is 1 to about 20. In some embodiments, m is 1 to 10, 1 to 7, 1 to 5,or 1 to 4.

In some embodiments, R₁ and R2 are both methoxy (—OMe).

In some embodiments, the nemorubicin component of anemorubicin-containing ADC is PNU-159682. In some such embodiments, thedrug portion of the ADC may have one of the following structures:

wherein the wavy line indicates the attachment to the linker (L).

Anthracyclines, including PNU-159682, may be conjugated to antibodiesthrough several linkage sites and a variety of linkers (US 2011/0076287;WO2009/099741; US 2010/0034837; WO 2010/009124), including the linkersdescribed herein.

Exemplary ADCs comprising a nemorubicin and linker include, but are notlimited to:

wherein:

R₁ and R2 are independently selected from H and C₁-C₆ alkyl; and

The linker of PNU-159682 maleimide acetal-Ab is acid-labile, while thelinkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab,and PNU-159682-val-cit-PAB-spacer(R¹R²)-Ab are protease cleavable.

(6) Other Drug Moieties

Drug moieties also include geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791); and enzymatically active toxins and fragments thereof,including, but not limited to, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, e.g., WO 93/21232.

Drug moieties also include compounds with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, ¹²⁵I, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. In some embodiments, when an immunoconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example Tc⁹⁹ or I¹²³, or a spin label for nuclear magnetic resonance(NMR) imaging (also known as magnetic resonance imaging, MRI), such aszirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents andconjugated to antibodies, e.g., for PET imaging (WO 2011/056983).

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, a peptide may be biosynthesized or chemicallysynthesized using suitable amino acid precursors comprising, forexample, one or more fluorine-19 atoms in place of one or morehydrogens. In some embodiments, labels such as Tc⁹⁹, I¹²³, Re¹⁸⁶, Re¹⁸⁸and In¹¹¹ can be attached via a cysteine residue in the antibody. Insome embodiments, yttrium-90 can be attached via a lysine residue of theantibody. In some embodiments, the IODOGEN method (Fraker et al (1978)Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporateiodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRCPress 1989) describes certain other methods.

In certain embodiments, an immunoconjugate may comprise an antibodyconjugated to a prodrug-activating enzyme. In some such embodiments, aprodrug-activating enzyme converts a prodrug (e.g., a peptidylchemotherapeutic agent, see WO 81/01145) to an active drug, such as ananti-cancer drug. Such immunoconjugates are useful, in some embodiments,in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymesthat may be conjugated to an antibody include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases,such as serratia protease, thermolysin, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; β-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. In some embodiments, enzymes may be covalently bound toantibodies by recombinant DNA techniques well known in the art. See,e.g., Neuberger et al., Nature 312:604-608 (1984).

c) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments above, an antibodymay have only one or several cysteine thiol groups, or may have only oneor several sufficiently reactive thiol groups through which a linker maybe attached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the average drug loading for an ADC ranges from 1 to about8; from about 2 to about 6; or from about 3 to about 5. Indeed, it hasbeen shown that for certain ADCs, the optimal ratio of drug moieties perantibody may be less than 8, and may be about 2 to about 5 (U.S. Pat.No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, and for example, by: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent, then theresulting product is a mixture of ADC compounds with a distribution ofone or more drug moieties attached to an antibody. The average number ofdrugs per antibody may be calculated from the mixture by a dual ELISAantibody assay, which is specific for antibody and specific for thedrug. Individual ADC molecules may be identified in the mixture by massspectroscopy and separated by HPLC, e.g. hydrophobic interactionchromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design &Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K. J., et al. “Effect of drug loading on thepharmacology, pharmacokinetics, and toxicity of an anti-CD30antibody-drug conjugate,” Abstract No. 624, American Association forCancer Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling thelocation of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certainembodiments, a homogeneous ADC with a single loading value may beisolated from the conjugation mixture by electrophoresis orchromatography.

d) Certain Methods of Preparing Immunoconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in U.S. Pat. No. 7,498,298, whichis expressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may also be introduced into anantibody by introducing one, two, three, four, or more cysteine residues(e.g., by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Exemplary nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Nonlimiting exemplary cross-linker reagents that may be used to prepareADC are described herein in the section titled “Exemplary Linkers.”Methods of using such cross-linker reagents to link two moieties,including a proteinaceous moiety and a chemical moiety, are known in theart. In some embodiments, a fusion protein comprising an antibody and acytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. A recombinant DNA molecule may comprise regions encoding theantibody and cytotoxic portions of the conjugate either adjacent to oneanother or separated by a region encoding a linker peptide which doesnot destroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-CD33 antibodies provided hereinis useful for detecting the presence of CD33 in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. A “biological sample” comprises, e.g., a cell or tissue(e.g., biopsy material, including cancerous or potentially cancerouslymphoid tissue, such as lymphocytes, lymphoblasts, monocytes,myelomonocytes, and mixtures thereof).

In one embodiment, an anti-CD33 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of CD33 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-CD33 antibody as described herein under conditionspermissive for binding of the anti-CD33 antibody to CD33, and detectingwhether a complex is formed between the anti-CD33 antibody and CD33 inthe biological sample. Such method may be an in vitro or in vivo method.In one embodiment, an anti-CD33 antibody is used to select subjectseligible for therapy with an anti-CD33 antibody, e.g. where CD33 is abiomarker for selection of patients. In a further embodiment, thebiological sample is a cell or tissue.

In a further embodiment, an anti-CD33 antibody is used in vivo todetect, e.g., by in vivo imaging, a CD33-positive cancer in a subject,e.g., for the purposes of diagnosing, prognosing, or staging cancer,determining the appropriate course of therapy, or monitoring response ofa cancer to therapy. One method known in the art for in vivo detectionis immuno-positron emission tomography (immuno-PET), as described, e.g.,in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel etal., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method isprovided for detecting a CD33-positive cancer in a subject, the methodcomprising administering a labeled anti-CD33 antibody to a subjecthaving or suspected of having a CD33-positive cancer, and detecting thelabeled anti-CD33 antibody in the subject, wherein detection of thelabeled anti-CD33 antibody indicates a CD33-positive cancer in thesubject. In certain of such embodiments, the labeled anti-CD33 antibodycomprises an anti-CD33 antibody conjugated to a positron emitter, suchas ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, 86Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In a particularembodiment, the positron emitter is ⁸⁹Zr.

In further embodiments, a method of diagnosis or detection comprisescontacting a first anti-CD33 antibody immobilized to a substrate with abiological sample to be tested for the presence of CD33, exposing thesubstrate to a second anti-CD33 antibody, and detecting whether thesecond anti-CD33 is bound to a complex between the first anti-CD33antibody and CD33 in the biological sample. A substrate may be anysupportive medium, e.g., glass, metal, ceramic, polymeric beads, slides,chips, and other substrates. In certain embodiments, a biological samplecomprises a cell or tissue. In certain embodiments, the first or secondanti-CD33 antibody is any of the antibodies described herein.

Exemplary disorders that may be diagnosed or detected according to anyof the above embodiments include CD33-positive cancers, such asCD33-positive AML, CD33-positive CML, CD33-positive MDS, CD33-positivechronic myelomonocytic leukemia, CD33-positive APL, CD33-positivechronic myeloproliferative disorder, CD33-positive thrombocyticleukemia, CD33-positive pre-B-ALL, CD33-positive preT-ALL, CD33-positivemultiple myeloma, CD33-positive mast cell disease, CD33-positive mastcell leukemia, CD33-positive mast cell sarcoma, CD33-positive myeloidsarcomas, CD33-positive lymphoid leukemia, and CD33-positiveundifferentiated leukemia. In some embodiments, a CD33-positive canceris a cancer that receives an anti-CD33 immunohistochemistry (IHC) or insitu hybridization (ISH) score greater than “0,” which corresponds tovery weak or no staining in >90% of tumor cells, under the conditionsdescribed herein in Example B. In another embodiment, a CD33-positivecancer expresses CD33 at a 1+, 2+ or 3+ level, as defined under theconditions described herein in Example B. In some embodiments, aCD33-positive cancer is a cancer that expresses CD33 according to areverse-transcriptase PCR (RT-PCR) assay that detects CD33 mRNA. In someembodiments, the RT-PCR is quantitative RT-PCR.

In certain embodiments, labeled anti-CD33 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, -galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like. In anotherembodiment, a label is a positron emitter. Positron emitters include butare not limited to ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In aparticular embodiment, a positron emitter is ⁸⁹Zr.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-CD33 antibody or immunoconjugateas described herein are prepared by mixing such antibody orimmunoconjugate having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody or immunoconjugate formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous antibody orimmunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-CD33 antibodies or immunoconjugates provided herein maybe used in methods, e.g., therapeutic methods.

In one aspect, an anti-CD33 antibody or immunoconjugate provided hereinis used in a method of inhibiting proliferation of a CD33-positive cell,the method comprising exposing the cell to the anti-CD33 antibody orimmunoconjugate under conditions permissive for binding of the anti-CD33antibody or immunoconjugate to CD33 on the surface of the cell, therebyinhibiting the proliferation of the cell. In certain embodiments, themethod is an in vitro or an in vivo method. In further embodiments, thecell is a lymphocyte, lymphoblast, monocyte, or myelomonocyte cell.

Inhibition of cell proliferation in vitro may be assayed using theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.). That assay determines the numberof viable cells in culture based on quantitation of ATP present, whichis an indication of metabolically active cells. See Crouch et al. (1993)J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al. (1995) Anti CancerDrugs 6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

In another aspect, an anti-CD33 antibody or immunoconjugate for use as amedicament is provided. In further aspects, an anti-CD33 antibody orimmunoconjugate for use in a method of treatment is provided. In certainembodiments, an anti-CD33 antibody or immunoconjugate for use intreating CD33-positive cancer is provided. In certain embodiments, theinvention provides an anti-CD33 antibody or immunoconjugate for use in amethod of treating an individual having a CD33-positive cancer, themethod comprising administering to the individual an effective amount ofthe anti-CD33 antibody or immunoconjugate. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow.

In a further aspect, the invention provides for the use of an anti-CD33antibody or immunoconjugate in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment ofCD33-positive cancer. In a further embodiment, the medicament is for usein a method of treating CD33-positive cancer, the method comprisingadministering to an individual having CD33-positive cancer an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below.

In a further aspect, the invention provides a method for treatingCD33-positive cancer. In one embodiment, the method comprisesadministering to an individual having such CD33-positive cancer aneffective amount of an anti-CD33 antibody or immunoconjugate. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below.

A CD33-positive cancer according to any of the above embodiments may be,e.g., CD33-positive AML, CD33-positive CML, CD33-positive MDS,CD33-positive chronic myelomonocytic leukemia, CD33-positive APL,CD33-positive chronic myeloproliferative disorder, CD33-positivethrombocytic leukemia, CD33-positive pre-B-ALL, CD33-positive preT-ALL,CD33-positive multiple myeloma, CD33-positive mast cell disease,CD33-positive mast cell leukemia, CD33-positive mast cell sarcoma,CD33-positive myeloid sarcomas, CD33-positive lymphoid leukemia, andCD33-positive undifferentiated leukemia. In some embodiments, aCD33-positive cancer is a cancer that receives an anti-CD33immunohistochemistry (IHC) or in situ hybridization (ISH) score greaterthan “0,” which corresponds to very weak or no staining in >90% of tumorcells, under the conditions described herein in Example B. In anotherembodiment, a CD33-positive cancer expresses CD33 at a 1+, 2+ or 3+level, as defined under the conditions described herein in Example B. Insome embodiments, a CD33-positive cancer is a cancer that expresses CD33according to a reverse-transcriptase PCR (RT-PCR) assay that detectsCD33 mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR.

An “individual” according to any of the above embodiments may be ahuman.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-CD33 antibodies or immunoconjugate providedherein, e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the anti-CD33antibodies or immunoconjugates provided herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of the anti-CD33 antibodies or immunoconjugates providedherein and at least one additional therapeutic agent, e.g., as describedbelow.

Antibodies or immunoconjugates of the invention can be used either aloneor in combination with other agents in a therapy. For instance, anantibody or immunoconjugate of the invention may be co-administered withat least one additional therapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or immunoconjugate of the invention canoccur prior to, simultaneously, and/or following, administration of theadditional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of antibody orimmunoconjugate, the severity and course of the disease, whether theantibody or immunoconjugate is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody or immunoconjugate, and the discretion ofthe attending physician. The antibody or immunoconjugate is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 jag/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or immunoconjugate can be aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody orimmunoconjugate would be in the range from about 0.05 mg/kg to about 10mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg (or any combination thereof) may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using both an immunoconjugate of theinvention and an anti-CD33 antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thedisorder and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or immunoconjugate of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody or immunoconjugate of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution ordextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1

A. Monoclonal Antibody Generation

Monoclonal antibodies against human (hu) and cynomolgus (cyno) CD33 weregenerated using the following procedures by immunizing animals withrecombinant hu and cyno CD33 extracellular domain (ECD, amino acids of1-262 huCD33 and 1-257 cynoCD33) fused to a C-terminal Flag (RADYKDDDDK)(SEQ ID NO: 124) expressed in a mammalian expression system.

Positive clones were expanded and re-screened for binding to huCD33 andcynoCD33 by ELISA and FACS. Nine clones were identified: 33H4, 33F9,27C6, 2E4, 7A1, 9C2, 9C3, 10D3 and 15G15 that reacted strongly byfluorescent activated cell sorting (FACs) with stable cell linesexpressing recombinant human and cynomolgus monkey CD33, and withtumor-derived CD33 expressed on Acute Myeloid Leukemia tumor cell lines.Variants were made of 9C3 and 15G15 including 9C3.2, 9C3.3, 9C3.4,15G15.33, 15G15.37, 15G15.83, 15G15.88, 15G15.7, 15G15.17, 15G15.30,15G15.31, 15G15.39, and 15G15.84. In some instances, monovalent bindingaffinities were determined by Biacore (data not shown).

Sequences of isolated heavy and light chains are found in FIGS. 1-4 andsequence listing below. Residue numbers are according to Kabat et al.,Sequences of proteins of immunological interest, 5th Ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991).

B. Species Cross-Reactivity

Monoclonal antibodies were tested to determine if they cross-react withcynoCD33 (which is 86.3% identical to huCD33 protein). HEK293AD cellsstably expressing human or cynomolgus monkey CD33 were used to determinespecies-specificity by FACS. Cells were incubated with antibody clonesat 1 jag/ml for 40 minutes at 4° C., washed and detected with either agoat-anti-mIgG (H+L) F(ab′)₂-488 or goat-anti-hIgG (H+L) F(ab′)₂-488secondary antibody. FIG. 5A-D shows that 6 antibodies (7A1, 9C2, 10D3,15G15, 27C6 and 33F9) recognized both recombinant hu and cynoCD33, whiletwo antibodies (33H4 and 23E4) had similar binding profiles to MY9.6,binding only to human CD33. Further confirmation of cross-reactivity tocynomolgus monkey CD33 was done by FACS analyses of blood fromcynomolgus (Mauritian origin). FIG. 7A-D shows 7A1, 9C2, 10D3, 15G15,33F9 and 27C6 stain cynomolgus CD14+/CD33⁺ myeloid cells, but 33H4, 23E4or MY9.6 did not. Antibody binding was also confirmed for humanCD14⁺/CD33⁺ myeloid cells. Tumor cells have the potential to alter theglycosylation pattern of proteins, for example, to escape from an immuneresponse, so to insure that our antibodies would not be affected by thismodification, FACS was done on AML tumor cell lines Molm13, HL-60,EOL-1, THP-1 and U937, and bone marrow from patients with confirmedcases of AML. FIG. 6A-D show a representative example of antibodiesbinding to Molm-13 or to a positive AML sample. These result suggeststhat antibody binding to CD33 is not affected by altered glycosylationfound in AML tumor cell lines or patient samples.

C. Monoclonal Antibody Epitope Grouping

Epitope binning of anti-CD33 antibodies was performed using the OctetRED384 instrument (ForteBio). Biotinylated CD33 was captured ontoStreptavidin biosensors at 10 jag/ml for 60 seconds. Binding of thefirst antibody to saturation was achieved by adding 50 jag/ml for 600seconds. The same biosensors were dipped into the competing antibodiesat 5 jag/ml and binding was measured for 300 seconds. The failure of thesecond antibody to bind in the presence of saturating quantities of thefirst antibody indicates the two antibodies were in the same epitopebin; the success of the second antibody to bind in the presence of thesaturating quantities of the first antibody indicates the two antibodieswere in different epitope bins. My9.6 was used as the first saturatingantibody, followed by competing antibodies 27C6, 23E4, 33H4, 33F9, 9C2,7A1, 10D3, and 15G15. Subsequent experiments used antibodies 33H4, 27C6,23E4, 7A1, 33F9, and 15G15 as the saturating antibody to complete andconfirm the analysis (data not shown).

FIG. 8A-C shows epitope binning of the antibodies to CD33, and showsthat 33F9, 7A1, 9C2, 10D3 and 15G15 bin with MY9.6, but 27C6, 23E4 and33H4 do not. It was also determined that 27C6 has a different epitopefrom all other antibodies, and 23E4 and 33H4 share an overlappingepitope (data not shown). Epitope binning to cyno-CD33 shows that 7A1,9C2, 10D3, and 15G15 bin together, but this bin does not include 27C6and 33F9 (FIG. 8D). This suggests 27C6 and 33F9 bind to a differentepitope on cyno-CD33. Data also revealed that 27C6 and 33F9 do not bintogether (data not shown). Although the human binning showed overlap ofMY9.6 with 7A1, 9C2, 10D3, 15G15 and 33F9, FACS data has shown thatMY9.6, 23E4 and 33H4 do not bind cyno-CD33, therefore the epitope ofMY9.6 to CD33 is probably not identical to 7A1, 9C2, 10D3, 15G15 and33F9.

Epitope grouping was also determined using a cell-based competitionbinding FACS assay. HEK293AD cells expressing recombinant human CD33were simultaneously incubated with a Dylight-650 labeled tracer antibody(0.3-1 jag/ml) and 50-100 fold excess of unlabeled competitor antibody.When the tracer is displaced by unlabeled antibody, competition hasoccurred indicating that the antibody binds to the same or similarregion on CD33—this should occur when the same antibody is used astracer and competitor. When there is no displacement of tracer by adifferent unlabeled antibody, the unlabeled antibody is binding to adifferent region in CD33.

FIG. 9 shows a representative example using 15G15 Dylight-650 tracerantibody with unlabeled competitor antibody at 50-fold excess of thetracer. As observed with Octet data, 27C6 did not compete with 15G15.Three other antibodies (9C2, 33F9, and 10D3) showed competition but notto the extent seen with unlabeled 15G15, suggesting that their epitopesmay be similar, but not identical.

As shown in FIG. 10A-B, 9C3 was shown to bind to hu and cynoCD33.However the competitor antibody, 15G15, failed to displace the 9C3labeled tracer antibody suggesting it binds to a different region onhuCD33 (FIG. 10C). Physical characterization of 9C3 identified anatypical N-linked glycosylation site in the heavy chain between CDR2 andCDR3 at position 69 (kabat #). Site-directed mutagenesis was used toremove the site and FIG. 11 shows an improvement in binding to CD33 byFACS and Scatchard Analysis (Table 2).

To determine whether the CD33 antibodies bind to either the Ig-like V orIg-like C domain of CD33, chimeric Ig-like domain membrane proteins wereengineered that contain either a CD33 Ig-like V (M17-V136 includingspacer H137-H143) linked to an irrelevant Ig-like C (including TM/CD;construct-88B) or an irrelevant Ig-like V linked to a CD33 Ig-like C(R144-Q228 including TM/CD L229) (construct-88) using standard molecularcloning methods. See FIG. 12A. N-terminal or cytoplasmic tags wereattached to confirm that 293 cells transfected with these constructsexpress protein on the cell membrane (data not shown). Briefly, 293cells were transiently transfected with constructs 88 & 88B usingpolyfect. After 48 hours, the cells were stained with 1-10 g/ml ofDylight-650 labeled 7A1, 9C2, 10D3 or 15G15 for 30-40 minutes at 4° C.,washed and analyzed on a BD FACS calibur.

In FIG. 12D, antibodies 7A1, 9C2, 10D3 and 15G5 showed significantbinding to the CD33 Ig-like V in construct 88B—at least 100 fold morecompared to the isotype control. However, there was no binding to theCD33 Ig-like C in construct 88 (FIG. 12C)—in fact binding was equivalentto mock transfected cells (FIG. 12B). A positive signal was detected inconstruct 88 by an antibody to the irrelevant Ig-like V domainconfirming that the construct was expressed on the cell surface (datanot shown).

The Ig-like V domain of CD33 contains two N-linked glycosylation sites(NXS/T) and a SNP at position 69 of CD33 (R69G; r2455069) that mayaffect binding of an antibody to CD33. To test the effects of the twoN-linked glycosylation sites, the serine residues at position S102 andS115 were substituted with alanine using standard site-specificmutagenesis (QuikChange II, Agilent Technologies) to reduce or abolishN-linked glycosylation at positions N100 and N113, respectively, in theIg-like V domain of a full-length huCD33 membrane construct. Constructscontained either a single mutation (S115A) or a double mutation(S102A/S115A) and were expressed in 293AD cells by transienttransfection using Polyfect (Promega) (FIG. 13A). FACS using 1 μg/ml ofantibody conjugated to Dylight-650 was done 48 hours later. FIG. 13Bshows the results of a representative example, clone 15G15, whichexhibited significant binding to transiently transfected cellsexpressing either the partially or fully deglycosylated Ig-like V formsof huCD33—as shown by the 18-44 fold higher fluorescence compared to theisotype control. The experiment demonstrates that binding of theantibodies is independent of N-linked glycosylation in the Ig-like Vdomain. (HEK 293AD stably expressing high levels of rhCD33 was used as apositive control stain and is not suitable as reference for quantitationof expression by the transiently transfected cell.)

The influence of SNP (R69G) on antibody binding to the Ig-like V domainwas investigated by the effect of glycine and arginine at amino acidposition number 69 in huCD33 using standard site-specific mutagenesisand expressing the R69G variant in 293AD cells as described above (FIG.14A).

FIG. 14B-C shows that the antibodies tested bound to the R69 CD33 andG69 CD33, and that binding was similar between the two forms of huCD33(data not shown).

D. Antibody Affinities

Scatchard analysis was performed following standard procedures (Holmeset al., Science 256:1205-1210 (1992)) to determine the relative bindingaffinities of the antibodies including 33H4, 33F9, 27C6, 2E4, 7A1, 9C2,9C3, 9C3.2, 9C3.3, 9C3.4, 10D3, 15G15, 15G15.33, 15G15.83, and 15G15.88.

Anti-CD33 antibodies were [I¹²⁵] labeled using the indirect lodogenmethod. The [I¹²⁵] labeled anti-CD33 antibodies were purified from free¹²⁵I-Na by gel filtration using a NAP-5 column (GE Healthcare); thepurified iodinated anti-CD33 antibodies had a range of specificactivities of 8-10 μCi/μg. Competition assay mixtures of 50 μL volumecontaining a fixed concentration of [I¹²⁵] labeled antibody anddecreasing concentrations of serially diluted, unlabeled antibody wereplaced into 96-well plates. HEK293AD cells stably expressing recombinanthu or cynoCD33 or Molm-13 tumor cells expressing endogenous CD33 werecultured in growth media at 37° C. in 5% CO₂. Cells were detached fromthe flask using Sigma Cell Dissociation Solution and were washed withbinding buffer, which consisted of Dulbecco's Modified Eagle Medium(DMEM) with 1% bovine serum albumin (BSA), 300 mM human IgG and 0.1%sodium azide. The washed cells were added to the 96 well plates at adensity of 100,000 cells in 0.2 mL of binding buffer. The finalconcentration of the [I¹²⁵] labeled antibody in each well was -250 μM.The final concentration of the unlabeled antibody in the competitionassay ranged from 1000 nM through ten 2-fold dilution steps to a 0 nMbuffer-only assay. Competition assays were carried out in triplicate.Competition assays were incubated for 2 hours at room temperature. Afterthe 2-hour incubation, the competition assays were transferred to aMillipore Multiscreen filter plate (Billerica, Mass.) and washed 4 timeswith binding buffer to separate the free from bound [I¹²⁵] labeledantibody. The filters were counted on a Wallac Wizard 1470 gamma counter(PerkinElmer Life and Analytical Sciences Inc.; Wellesley, Mass.). Thebinding data was evaluated using NewLigand software (Genentech), whichuses the fitting algorithm of Munson and Robard to determine the bindingaffinity of the antibody (Munson and Robard 1980).

Table 2 shows the affinity (kD range of 0.2-23 nM) to recombinant hu andcynoCD33 expressed by HEK293AD CD33 stable cells and to Molm-13 cells.

TABLE 2 Antibody Affinity [kD = nM] to CD33 (Scatchard Analysis) AB ID293-huCD33 293-cynCD33 Molm-13 2E3 2.5 X 3.0 33H4 2.4 X 0.6 27C6 13.4 4.5 23 33F9 8.7  0.92 92 7A1 3.0  0.54 6.3 9C2 6.5 0.7 10.3 9C3 0.8 0.65.8 9C3.2 ND ND 2.0 9C3.3 ND ND 2.2 9C3.4 ND ND 2.7 10D3 3.3 0.7 2.615G15 1.3 0.3 4.8 15G15.33 0.7 0.3 0.9 15G15.83 0.5 0.2 0.5 15G15.88 0.70.2 0.8

E. Internalization of Anti-CD33 Antibody

One desirable attribute of an ADC target is the ability to internalizethe antibody into a degradative compartment in the cell. To determinewhether anti-CD33 antibody gets internalized upon binding, HL-60 orMolm-13 cells were pre-incubated for 2 hours at 37° C. with 0.3 mg/mlhIgG in RPMI medium to reduce non-specific binding to FcR before seedingin cell culture treated 4-well chamber slides (Nalge NuncInternational). Antibody directly conjugated to Dylight 488 at a finalconcentration of 1 μg/mL was incubated with hIgG-blocked cells on icefor 30 minutes in the dark. The cells were immediately imaged to showmembrane staining (T0) and followed with time-lapsed photography over a10 hour period at 37° C. with a Leica SP5 confocal microscope. As shownin FIG. 15A, a representative example, 15G15, is rapidly internalizedwithin 30 minutes by HL-60 cells. Localization of 15G15 to the lysosomewas confirmed using an in vitro cell-based assay measuring the abilityof an antibody drug conjugate to kill target cells. Briefly, Molm-13 orEOL-1 cells were pre-incubated with RPMI containing 0.3 mg/ml lowendotoxin hIgG for 2 hours at 37° C. to reduce non-specific binding toFcR, and plated at a density of 8,000-16,000 cells/well in a 96-plate.The test articles, isotype-L-D#1 or 15G15.33 L-D#1, were added to thecells with a final concentration range of 0-1000 ng/ml in 3-fold stepsand incubated for ˜60 hours at 37° C. with 5% CO₂. Cell viability wasdetermined using CellTiter-Glo (Promega, Inc) and an Envision 2012Multilabel Reader (Perkin Elmer). FIG. 15B shows specific killing andcomplete ablation of target cells with 15G15.33-L-D#1 ADC compared tothe isotype ADC (EC₅₀ of 6 and 63 ng/ml respectively), thus confirmingADC trafficking and processing in the lysosome. Both ADC's had a similardrug load.

F. Production of Anti-CD33 Antibody Drug Conjugates

For larger scale antibody production, antibodies were produced in CHOcells. Vectors coding for VL and VH were transfected into CHO cells andIgG was purified from cell culture media by protein A affinitychromatography.

Anti-CD33 antibody-drug conjugates (ADCs) were produced by conjugating15G15 with a heavy chain A118C mutation (15G15 thio-HC A118C) and15G15.33 with a heavy chain A118C mutation (15G15.33 thio-HC A118C) or alight chain V205C mutation (15G15.33 thio-LC V205C) to the drug-linkermoiety monomethyl-pyridyl disulfide, N10-linked pyrrolobenzodiazepine(see FIG. 16; L-D #1) or maleimide with acetal linker-PNU (see FIG. 17;L-D #2). As initially isolated, the engineered cysteine residues inantibodies 15G15 and 15G15.33 exist as mixed disulfides with cellularthiols (e.g., glutathione) and are thus unavailable for conjugation.Partial reduction of these antibodies (e.g., with DTT), purification,and reoxidation with dehydroascorbic acid (DHAA) gives antibodies withfree cysteine sulfhydryl groups available for conjugation, as previouslydescribed, e.g., in Junutula et al. (2008) Nat. Biotechnol. 26:925-932and US 2011/0301334. Briefly, the antibodies were combined with thedrug-linker moiety to allow conjugation of the drug-linker moiety to thefree cysteine residues of the antibody. After several hours, the ADCswere purified. The drug load (average number of drug moieties perantibody) for each ADC was determined and was between 1.4-1.6 for thePBD conjugates and 1.3 for the PNU conjugates.

G. Efficacy of Anti-CD33 Antibody Drug Conjugates in HL-60 and EOL-1Human Acute Myeloid Leukemia Cell Line Xenograft Models

The efficacy of the anti-CD33 ADCs was investigated using human AcuteMyeloid Leukemia xenograft models, HL-60 (AML subtype M2) and EOL-1 (AMLsubtype M4a). Both are associated with intermediate to poor prognosis asa result of their genetics and molecular aberrations. Female C.B-17 SCIDmice (Charles River Laboratories; Hollister, Calif.) were eachinoculated subcutaneously in the flank area with five million cells ofHL-60 or EOL-1. When the xenograft tumors reached an average tumorvolume of 100-300 mm³ (referred to as Day 0), animals were randomizedinto groups of 7-10 mice each and received a single intravenousinjection of the ADCs. Approximately 4 hours prior to administration ofADCs, animals were dosed intraperitoneally with excess amount (30 mg/kg)of anti-gD control antibody to block possible nonspecific antibodybinding sites on the tumor cells. Tumors and body weights of mice weremeasured 1-2 times a week throughout the study. Mice were promptlyeuthanized when body weight loss was >20% of their starting weight. Allanimals were euthanized before tumors reached 3000 mm3 or showed signsof impending ulceration. The presence of the antibodies was confirmed byPK bleeds at 1, 7 and 14 days post injection.

As shown in FIG. 16A-B, substantial tumor growth inhibition was achievedin both the HL-60 and EOL-1 models at the 1 mg/kg or 20 μg/m² dose of15G15.33-L-D#1, while lower doses resulted in retarded tumor growthcompared to the negative control antibody-drug conjugate. As shown inFIG. 17, a single 11.9 mg/kg dose of 15G15 L-D #2 was found to retardtumor growth in the HL-60 model.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Table of Sequences SEQ NAME SEQUENCE ID NO Human CD33MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY 1 (UniProt No.DKNSPVHGYW FREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN 20138)CSLSIVDARR RDNGSYFFRM ERGSTKYSYK SPQLSVHVTD LTHRPKILIPGTLEPGHSKN LTCSVSWACE QGTPPIFSWL SAAPTSLGPR TTHSSVLIITPRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTT GIFPGDGSGKQETRAGVVHG AIGGAGVTAL LALCLCLIFF IVKTHRRKAA RTAVGRNDTHPTTGSASPKH QKKSKLHGPT ETSSCSGAAP TVEMDEELHY ASLNFHGMNP SKDTSTEYSE VRTQIg-like V- PNFWLQVQES VTVQEGLCVL VPCTFFHPIP YYDKNSPVHG YWFREGAIIS 2type domain RDSPVATNKL DQEVQEETQG RFRLLGDPSR NNCSLSIVDA RRRDNGSYFF(amino acids RMERGSTKYS YKSPQLS 19-135 of full length CD33) Ig-like C-PKILIPGTLE PGHSKNLTCS VSWACEQGTP PIFSWLSAAP TSLGPRTTHS 3 type domainSVLIITPRPQ DHGTNLTCQV KFAGAGVTTE RTIQ (amino acids 145-228 offull length CD33) Cyno CD33MPLLLLPLLWAGALAMDPRVRLEVQESVTVQEGLCVLVPCTFFHPVPYHTRNSPVH 4GYWFREGAIVSLDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMEKGSTKYSYKSTQLSVHVTDLTHRPQILIPGALDPDHSKNLTCSVPWACEQGTPPIFSWMSAAPTSLGLRTTHSSVLIITPRPQDHGTNLTCQVKFPGAGVTTERTIQLNVSYASQNPRTDIFLGDGSGKQGVVQGAIGGAGVTVLLALCLCLIFFTVKTHRRKAARTAVGRIDTHPATGPTSSKHQKKSKLHGATETSGCSGTTLTVEMDEELHYASLNFHGMNPSEDTSTEYSEVRTQ 7A1, 9C2, RSSQSLLHSNGYNYLD 5 10D3, 15G15,15G15.33, 15G15.37, 15G15.83, 15G15.88, 15G15.7, 15G15.17, 15G15.30,15G15.31, 15G15.39, 15G15.84-HVR L1 7A1, 9C2, LGSNRAS 6 10D3, 15G15,15G15.7, 15G15.17, 15G15.30, 15G15.31, 15G15.39, 15G15.84-HVR L27A1, 9C2, MQALQTPWT 7 10D3, 15G15, 15G15.33, 15G15.37, 15G15.83,15G15.88, 15G15.7, 15G15.17, 15G15.30, 15G15.31, 15G15.39, 15G15.84-HVRL3 7A1-HVR H1 SYAVS 8 7A1-HVR H2 GIIPIFGTADYAQKFQG 9 7A1-HVR H3 ELADVFDI10 9C2-HVR H1 SYSIS 11 9C2-HVR H2 EIIPIFGTADYAQKFQG 12 9C2-HVR H3TWADAFDI 13 9C3-HVR L1 RASQGIRNDLG 14 9C3-HVR L2 AASSLQS 15 9C3-HVR L3LQHNSYPWT 16 9C3-HVR H1 GNYMS 17 9C3-HVR H2 LIYSGDSTYYADSVKG 189C3-HVR H3 DGYYVSDMVV 19 10D3-HVR H1 SHAIS 20 10D3-HVR H2GIIPIFGSANYAQKFQG 21 10D3-HVR H3 ELLDVFDI 22 15G15, NHAIS 23 15G15.33,15G15.37, 15G15.83, 15G15.88, 15G15.17, 15G15.30, 15G15.31, 15G15.39,15G15.84-HVR H1 15G15, GIIPIFGTANYAQKFQG 24 15G15.33, 15G15.37,15G15.83, 15G15.88, 15G15.7, 15G15.17- HVR H2 15G15, EWADVFDI 2515G15.33, 15G15.37, 15G15.83, 15G15.88, 15G15.7, 15G15.17, 15G15.30,15G15.31, 15G15.39, 15G15.84-HVR H3 15G15.33-HVR LGVNSVS 26 L215G15.37-HVR LGSHRDS 27 L2 15G15.83-HVR LGAYTVS 28 L2 15G15.88-HVRLGNYRVS 29 L2 15G15.7-HVR GHKVS 30 H1 15G15.17-HVR GIIPILGLDYYAQKFQG 31H2 15G15.30-HVR GIIPVLGYAYYAQKFQG 32 H2 15G15.31-HVR GIIPILGYAYYAQKFQG33 H2 15G15.39-HVR GIIPILGISYYAQKFQG 34 H2 15G15.84-HVRSIIPVIGYDYYAQKFQG 35 H2 23E4-HVR L1 RSSQTIVHSNGNTYLE 36 23E4-HVR L2KVSNRFS 37 23E4-HVR L3 FQGSHVPPT 38 23E4-HVR H1 NYWMN 39 23E4-HVR H2MIDPSDNETHYSQMFKD 40 23E4-HVR H3 YYGNFGWFVY 41 27C6-HVR L1 KASQDVGDAVA42 27C6-HVR L2 WTSTRHT 43 27C6-HVR L3 QQYRSTPLT 44 27C6, 33F3- SYNMY 45HVR H1 27C6-HVR H2 YIDPYNGGTRHNQKFKD 46 27C6-HVR H3 QNYEYFDY 4733F3-HVR L1 KASQDVNTAVA 48 33F3-HVR L2 WASTRHT 49 33F3-HVR L3 QQHSGTPLT50 33F3-HVR H2 YIDPYNGGTSYNQKFKG Si 33F3-HVR H3 AAYFYFDY 52 33F9-HVR L1LASQTIGTWLA 53 33F9-HVR L2 AATTLAD 54 33F9-HVR L3 QQLYSTPLT 5533F9-HVR H1 SYVMH 56 33F9-HVR H2 YINPYNDGTKYNDKFKG 57 33F9-HVR H3GSNYEDFAMDY 58 33H4-HVR L1 RASESVDSYGNSYLH 59 33H4-HVR L2 LASNLES 6033H4-HVR L3 QQNNEDPWT 61 33H4-HVR H1 TFPIE 62 33H4-HVR H2NFHPYNDQTKYNEEFKG 63 33H4-HVR H3 GYYYAFDF 64 7A1 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 65LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK7A1 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGTFT SYAVSWVRQA PGQGLEWMGG 66IIPIFGTADY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCARELADVFDIWGQG TMVTVSS 9C2 V_(L)DVVMTQSPLS LPVAPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 67LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKLE IK9C2 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGDTFS SYSISWVRQA PGQGLEWMGE 68IIPIFGTADY AQKFQGRVTI TADISTTTAY MELSSLRSED TAVYYCARTWADAFDIWGQG TMVTVSS 9C3 V_(L)DIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKRLIYA 69ASSLQSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ HNSYPWTFGQ GTKLEIK 9C3 V_(H)EVQLVESGGA LIQPGGSLRL SCVASGFTIS GNYMSWVRQA PGKGLEWVSL 70IYSGDSTYYA DSVKGRFNIS RDISKNTVYL QMNSLRVEDT AVYYCVRDGYYVSDMVVWGK GTTVTVSS 9C3.2 V_(L)DIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKRLIYA 71ASSLQSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ HNSYPWTFGQ GTKLEIK9C3.2 V_(H) EVQLVESGGA LIQPGGSLRL SCVASGFTIS GNYMSWVRQA PGKGLEWVSL 72IYSGDSTYYA DSVKGRFTIS RDISKNTVYL QMNSLRVEDT AVYYCVRDGYYVSDMVVWGK GTTVTVSS 9C3.3 V_(L)DIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKRLIYA 73ASSLQSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ HNSYPWTFGQ GTKLEIK9C3.3 V_(H) EVQLVESGGA LIQPGGSLRL SCVASGFTIS GNYMSWVRQA PGKGLEWVSL 74IYSGDSTYYA DSVKGRFSIS RDISKNTVYL QMNSLRVEDT AVYYCVRDGYYVSDMVVWGK GTTVTVSS 9C3.4 V_(L)DIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKRLIYA 75ASSLQSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ HNSYPWTFGQ GTKLEIK9C3.4 V_(H) EVQLVESGGA LIQPGGSLRL SCVASGFTIS GNYMSWVRQA PGKGLEWVSL 76IYSGDSTYYA DSVKGRFAIS RDISKNTVYL QMNSLRVEDT AVYYCVRDGYYVSDMVVWGK GTTVTVSS 10D3 V_(L)DVVMTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 77LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK10D3 V_(H) EVQLVESGAE VKKPGSSVKV SCKASGGTLI SHAISWVRQV PGQGLEWMGG 78IIPIFGSANY AQKFQGRVTI TADDSTNTAY LELSSLRSED TAVYYCARELLDVFDIWGQG TMVTVSS 15G15 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 79LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 80IIPIFGTANY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.33 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 81LLIYLGVNSV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.33 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 82IIPIFGTANY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.37 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 83LLIYLGSHRD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.37 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 84IIPIFGTANY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.83 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 85LLIYLGAYTV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.83 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 86IIPIFGTANY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.88 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 87LLIYLGNYRV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.88 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 88IIPIFGTANY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.7 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 89LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.7 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS GHKVSWVRQA PGQGLEWMGG 90IIPIFGTANY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.17 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 91LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.17 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 92IIPILGLDYY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.30 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 93LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.30 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 94IIPVLGYAYY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.31 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 95LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.31 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 96IIPILGYAYY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.39 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 97LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.39 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGG 98IIPILGISYY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 15G15.84 V_(L)EIVLTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ 99LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTP WTFGQGTKVE IK15G15.84 V_(H) QVQLVQSGAE VKKPGSSVKV SCKASGGIFS NHAISWVRQA PGQGLEWMGS100 IIPVIGYDYY AQKFQGRVTI TADESTSTAF MELSSLRSED TAVYYCAREWADVFDIWGQG TMVTVSS 23E4 V_(L)DIFMTQTPLS LPVSLGDPAS ISCRSSQTIV HSNGNTYLEW YLQKPGQSPK 101LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP PTFGGGTKVE IK23E4 V_(H) EVQLQQSGAE LVRPGASVKL SCKASGYTFT NYWMNWVKQR PGQGLEWIGM 102IDPSDNETHY SQMFKDKATL TVDKSSSTAY MQLISLTSED SAVYYCAGYYGNFGWFVYWG QGTLVTVSA 27C6 V_(L)DIVLTQSPKF MSTSVGDRVS ITCKASQDVG DAVAWYQQKP GQSPKLLFYW 103TSTRHTGVPD RFTGSGSGTE FTLTIRNVQS EDLADYFCQQ YRSTPLTFGS GTKVEIK27C6 V_(H) EVQLQQSGPE LVKPGASVKV SCKASGYAFT SYNMYWVKQS HGKSLEWIGY 104IDPYNGGTRH NQKFKDKATL TVDKSSSTAY MHLNSLTSED SAVYYCASQNYEYFDYWGQG TTLTVSS 33F3 V_(L)EIQMTQSPKF MSTSVGDRVS ITCKASQDVN TAVAWYQQKP GQSPKLLIYW 105ASTRHTGVPD RFTGSGSGTD YTLTISSVQA EDLALYYCQQ HSGTPLTFGA GTKVEIK33F3 V_(H) EVQLQQSGPE LVKPGASVKV SCKASGYAFT SYNMYWVKQS HGKSLEWIGY 106IDPYNGGTSY NQKFKGKATL TVDKSSSTAY MHLNSLTSED SAVYFCAPAAYFYFDYWGQG TTLTVSS 33F9 V_(L)DIVMTQSPAS QSASLGESVT ITCLASQTIG TWLAWYQQKP GKSPQLLIYA 107ATTLADGVPS RFSGSGSGTK FSFKISSLQA EDFVSYYCQQ LYSTPLTFGG GTKVEIK33F9 V_(H) EVQLQQSGPE LVKPGASVKM SCKASGYTFT SYVMHWMKQK PGQGLEWIGY 108INPYNDGTKY NDKFKGKATL TSDKSSSTAY MELSSLTSED SAVYYCARGSNYEDFAMDYR GQGTSVTVSS 33H4 V_(L)DIQMTQSPAS LTVSLGQRAT ISCRASESVD SYGNSYLHWY QQKPGQPPQL 109LIYLASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YCQQNNEDPW TFGGGTKVEI K33H4 V_(H) EVQLQQSGAE LVKPGASVKM SCKAFGYTFT TFPIEWMKQS HGKSLEWIGN 110FHPYNDQTKY NEEFKGRAKL TIDRSSSTVY LELGRLTSDD SAVYYCARGYYYAFDFWGQG TTLTVSS CON1-HVR L2LGX₁X₂X₃X₄S, wherein X₁ is S, V, A or N, X₂ is N, H, or Y, 111X₃ is R, S, or T, and X₄ is A, V, or D CON1-HVR H1X₅X₆X₇X₈S, wherein X₅ is S, N, or G, X₆ is Y or H, X₇ is A, 112S, or K, and X₈ is V or I CON1-HVR H2X₉IIPX₁₀X₁₁GX₁₂X₁₃X₁₄YAQKFQG, wherein X9 is G, E, or S, X₁₀ 113is I or V, X₁₁ is F, L, or I, X₁₂ is T, S, L, Y, or I, X₁₃is A, D, or S, and X₁₄ is D, N, or Y CON1-HVR H3X₁₅X₁₆X₁₇DX₁₈FDI, wherein X₁₅ is E or T, X₁₆ is L or W, X₁₇ 114is A or L, X₁₈ is V or A CON2-HVR H1X₁₉X₂₀X₂₁X₂₂S, wherein X₁₉ is S or N, X₂₀ is Y or H, X₂₁ is A 115or S, and X₂₂ is V or I CON2-HVR H2X₂₃IIPIFGX₂₄AX₂₅YAQKFQG, wherein X₂₃ is G or E, X₂₄ is T or 116S, X₂₅ is D or N CON2-HVR H3X₂₆X₂₇X₂₈DX₂₉FDI, wherein X₂₆ is E or T, X₂₇ is L or W, X₂₈ 117is A or L, X₂₉ is V or A CON3-HVR H1X₃₀HX₃₁X₃₂S, wherein X₃₀ is N or G, X₃₁ is A or K, and X₃₂ is 118 V or ICON3-HVR H2 X₃₁IIPX₃₂X₃₃GX₃₄X₃₅X₃₆YAQKFQG, wherein X₃₁ is G or S, 119X₃₂ is I or V, X₃₃ is F, L, or I, X₃₄ is T, L, Y, or I,X₃₅ is A, D, or S, and X14 is N or Y V205C cysteineTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW 120 engineered lightKVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK chain constantHKVYACEVTH QGLSSPCTKS FNRGEC region (Igκ) A118C cysteineCSTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS 121 engineeredWNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT heavy chainYICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG constant regionPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW (IgG1)YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

1.-51. (canceled)
 52. An isolated antibody that binds to CD33, whereinthe antibody comprises: (i) (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:39; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:40; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO:41; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:36;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:37; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:38; (ii) (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:45; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:46; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:47; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:42; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:43; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:44; (iii) (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:45; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:51; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:52; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:48; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:49; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:50; (iv) (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:56; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:57; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:58; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:53; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:54; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:55; or (v) (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:62; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:63; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:64; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:60; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:61; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:62.
 53. The antibody ofclaim 52, wherein the antibody comprises: a) a heavy chain variableregion comprising the sequence of SEQ ID NO: 102 and a light chainvariable region comprising the sequence of SEQ ID NO: 101; b) a heavychain variable region comprising the sequence of SEQ ID NO: 104 and alight chain variable region comprising the sequence of SEQ ID NO: 103;c) a heavy chain variable region comprising the sequence of SEQ ID NO:106 and a light chain variable region comprising the sequence of SEQ IDNO: 105; d) a heavy chain variable region comprising the sequence of SEQID NO: 108 and a light chain variable region comprising the sequence ofSEQ ID NO: 107; or e) a heavy chain variable region comprising thesequence of SEQ ID NO: 110 and a light chain variable region comprisingthe sequence of SEQ ID NO:
 109. 54. The antibody of claim 52, whereinthe antibody has one or more of the following characteristics: a) bindsto recombinant human CD33; b) binds to recombinant cynomolgus monkeyCD33; c) binds to endogenous CD33 on the surface of human peripheralblood mononucleocytes (PBMCs); d) binds to endogenous CD33 on thesurface of cynomolgus monkey PBMCs; e) binds to endogenous CD33 on thesurface of a cancer cell; f) binds to endogenous CD33 on the surface ofan AML cancer cell; g) binds to endogenous CD33 on the surface ofMolm-13 cells; h) binds to CD33 comprising a R69G mutation; i) binds toendogenous human CD33 with a Kd of less than 15 nM, less than 10 nM,less than 7 nM, less than 5 nM, or less than 3 nM; j) binds torecombinant human CD33 with a Kd of less than 10 nM, less than 7 nM,less than 5 nM, or less than 3 nM; and/or k) binds to recombinantcynomolgus monkey CD33 with a Kd of less than 10 nM, less than 7 nM,less than 5 nM, or less than 3 nM, less than 2 nM, or less than 1 nM.55. The antibody of claim 52, which is a monoclonal antibody.
 56. Theantibody of claim 52, which is a humanized or chimeric antibody.
 57. Theantibody of claim 52, which is an antibody fragment that binds CD33. 58.The antibody of claim 52, wherein CD33 is human CD33 comprising aminoacids 18 to 364 of SEQ ID NO:
 1. 59. The antibody of claim 52, which isan IgG1, IgG2a or IgG2b antibody.
 60. An isolated nucleic acid encodingthe antibody of claim
 52. 61. A host cell comprising the nucleic acid ofclaim
 60. 62. A method of producing an antibody comprising culturing thehost cell of claim 61 so that the antibody is produced.
 63. The methodof claim 62, further comprising isolating the antibody.
 64. Animmunoconjugate comprising the antibody of claim 52 and a cytotoxicagent.
 65. The immunoconjugate of claim 64 having the formula Ab-(L-D)p,wherein: (a) Ab is the antibody; (b) L is a linker; (c) D is a cytotoxicagent; and (d) p ranges from 1-8.
 66. The immunoconjugate of claim 64,wherein the cytotoxic agent is selected from a maytansinoid, acalicheamicin, a pyrrolobenzodiazepine, and a nemorubicin derivative.67. The immunoconjugate of claim 65, wherein D is apyrrolobenzodiazepine of Formula A:

wherein the dotted lines indicate the optional presence of a double bondbetween C1 and C2 or C2 and C3; R² is independently selected from H, OH,═O, ═CH₂, CN, R, OR, ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, andoptionally further selected from halo or dihalo, wherein R^(D) isindependently selected from R, CO₂R, COR, CHO, CO₂H, and halo; R⁶ and R⁹are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo; R⁷ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn and halo; Q is independently selectedfrom O, S and NH; R¹¹ is either H, or R or, where Q is O, SO₃M, where Mis a metal cation; R and R′ are each independently selected fromoptionally substituted C₁₋₈ alkyl, C₃₋₈ heterocyclyl and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring; R¹², R¹⁶, R¹⁹and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷ respectively; R″ is a C₃₋₁₂alkylene group, which chain may be interrupted by one or moreheteroatoms and/or aromatic rings that are optionally substituted; and Xand X′ are independently selected from O, S and N(H).
 68. Theimmunoconjugate of claim 65, wherein D has the structure:

wherein n is 0 or
 1. 69. The immunoconjugate of claim 65, wherein D is anemorubicin derivative.
 70. The immunoconjugate of claim 69, wherein Dhas a structure selected from:


71. The immunoconjugate of claim 65, wherein the linker is cleavable bya protease.
 72. The immunoconjugate of claim 65, wherein the linker isacid-labile.
 73. The immunoconjugate of claim 72, wherein the linkercomprises hydrazone.
 74. The immunoconjugate of claim 70 having aformula selected from:


75. The immunoconjugate of claim 65, wherein p ranges from 2-5.
 76. Apharmaceutical formulation comprising the immunoconjugate of claim 64and a pharmaceutically acceptable carrier.
 77. The pharmaceuticalformulation of claim 76, further comprising an additional therapeuticagent.
 78. A method of treating an individual having a CD33-positivecancer, the method comprising administering to the individual aneffective amount of the immunoconjugate of claim
 64. 79. The method ofclaim 78, wherein the CD33-positive cancer is AML.
 80. The method ofclaim 78, further comprising administering an additional therapeuticagent to the individual.
 81. A method of inhibiting proliferation of aCD33-positive cell, the method comprising exposing the cell to theimmunoconjugate of claim 64 under conditions permissive for binding ofthe immunoconjugate to CD33 on the surface of the cell, therebyinhibiting proliferation of the cell.
 82. The method of claim 81,wherein the cell is an AML cancer cell.
 83. The antibody of claim 52conjugated to a label.
 84. The antibody of claim 83, wherein the labelis a positron emitter.
 85. The antibody of claim 84, wherein thepositron emitter is ⁸⁹Zr.
 86. A method of detecting human CD33 in abiological sample comprising contacting the biological sample with theanti-CD33 antibody of claim 52 under conditions permissive for bindingof the anti-CD33 antibody to a naturally occurring human CD33, anddetecting whether a complex is formed between the anti-CD33 antibody anda naturally occurring human CD33 in the biological sample.
 87. Themethod of claim 86, wherein the biological sample is a AML endometrialcancer sample.
 88. A method for detecting a CD33-positive cancercomprising (i) administering a labeled anti-CD33 antibody to a subjecthaving or suspected of having a CD33-positive cancer, wherein thelabeled anti-CD33 antibody comprises the anti-CD33 antibody of claim 52,and (ii) detecting the labeled anti-CD33 antibody in the subject,wherein detection of the labeled anti-CD33 antibody indicates aCD33-positive cancer in the subject.
 89. The method of claim 88, whereinthe labeled anti-CD33 antibody comprises an anti-CD33 antibodyconjugated to a positron emitter.
 90. The method of claim 89, whereinthe positron emitter is ⁸⁹Zr.